Proteopedia - User contributions [en]

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-14T21:27:31Z

Luis E Ramirez-Tapia:

<Structure load='Sele.pdb' size='400' frame='true' align='right' caption='CheW CheA CheY' scene='User:Luis_E_Ramirez-Tapia/Sandbox_4/Initial/1' />

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Initial/1'>TextToBeDisplayed</scene>

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Initial/2'>TextToBeDisplayed</scene>

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-13T20:23:56Z

Luis E Ramirez-Tapia:

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-13T20:22:53Z

Luis E Ramirez-Tapia:

<Structure load='Sele.pdb' size='400' frame='true' align='right' caption='CheW CheA CheY' scene='Initial' />

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Initial/1'>TextToBeDisplayed</scene>

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-13T20:22:09Z

Luis E Ramirez-Tapia:

<Structure load='Sele.pdb' size='400' frame='true' align='right' caption='CheW CheA CheY' scene=Initial/1 />

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Initial/1'>TextToBeDisplayed</scene>

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-13T19:59:54Z

Luis E Ramirez-Tapia: Replacing page with '<Structure load='Sele.pdb' size='400' frame='true' align='right' caption='CheW CheA CheY' scene='Insert optional scene name here' />'

File:Sele.pdb

2011-12-13T19:58:34Z

Luis E Ramirez-Tapia: CheW CheY, CheA receptor from e.coli

== Summary ==
CheW CheY, CheA receptor from e.coli
== Licensing ==
{{self|cc-by-sa-3.0|GFDL}}

User:Luis E Ramirez-Tapia

2011-12-13T19:56:42Z

Luis E Ramirez-Tapia:

Ph.D student at Umass Amherst. Actually working with Professor Craig Martin in undestanding the mechanism of T7 RNA polymerase.

*[[user:Luis E Ramirez-Tapia/Sandbox 1]]
*[[user:Luis E Ramirez-Tapia/Sandbox 2]]
*[[user:Luis E Ramirez-Tapia/Sandbox 3]]
*[[user:Luis E Ramirez-Tapia/Sandbox 4]]
*[[user:Luis E Ramirez-Tapia/T7 RNA polymerase]]

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T18:16:49Z

Luis E Ramirez-Tapia:

<Structure load='1QOY' size='300' frame='true' align='right' caption='Insert caption here' scene='<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/5'>TextToBeDisplayed</scene>'/>

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/1'>Test</scene>

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya_1/1'>ClyA 1</scene>

{{Seed}}
[[Image:1qoy.png|left|200px]]


{{STRUCTURE_1qoy| PDB=1qoy | SCENE= }}

===E.COLI HEMOLYSIN E (HLYE, CLYA, SHEA)===


{{ABSTRACT_PUBMED_10660049}}

==About this Structure==
1QOY is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOY OCA].

==Reference==
E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy., Wallace AJ, Stillman TJ, Atkins A, Jamieson SJ, Bullough PA, Green J, Artymiuk PJ, Cell. 2000 Jan 21;100(2):265-76. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/10660049 10660049]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Artymiuk, P J.]]
[[Category: Atkins, A.]]
[[Category: Bullough, P A.]]
[[Category: Green, J.]]
[[Category: Jamieson, S J.]]
[[Category: Stillman, T J.]]
[[Category: Wallace, A J.]]
[[Category: Cytolysin]]
[[Category: Hemolysin]]
[[Category: Membrane pore former]]
[[Category: Pore]]
[[Category: Toxin]]

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:22:40 2008''

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T18:11:59Z

Luis E Ramirez-Tapia:

<Structure load='1QOY' size='300' frame='true' align='right' caption='Insert caption here' scene='<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/5'>TextToBeDisplayed</scene>'/>

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/1'>Test</scene>

{{Seed}}
[[Image:1qoy.png|left|200px]]


{{STRUCTURE_1qoy| PDB=1qoy | SCENE= }}

===E.COLI HEMOLYSIN E (HLYE, CLYA, SHEA)===


{{ABSTRACT_PUBMED_10660049}}

==About this Structure==
1QOY is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOY OCA].

==Reference==
E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy., Wallace AJ, Stillman TJ, Atkins A, Jamieson SJ, Bullough PA, Green J, Artymiuk PJ, Cell. 2000 Jan 21;100(2):265-76. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/10660049 10660049]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Artymiuk, P J.]]
[[Category: Atkins, A.]]
[[Category: Bullough, P A.]]
[[Category: Green, J.]]
[[Category: Jamieson, S J.]]
[[Category: Stillman, T J.]]
[[Category: Wallace, A J.]]
[[Category: Cytolysin]]
[[Category: Hemolysin]]
[[Category: Membrane pore former]]
[[Category: Pore]]
[[Category: Toxin]]

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:22:40 2008''

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T18:10:05Z

Luis E Ramirez-Tapia:

<Structure load='1QOY' size='500' frame='true' align='right' caption='Insert caption here' scene='<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/5'>TextToBeDisplayed</scene>'/>

<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/1'>Test</scene>

{{Seed}}
[[Image:1qoy.png|left|200px]]


{{STRUCTURE_1qoy| PDB=1qoy | SCENE= }}

===E.COLI HEMOLYSIN E (HLYE, CLYA, SHEA)===


{{ABSTRACT_PUBMED_10660049}}

==About this Structure==
1QOY is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOY OCA].

==Reference==
E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy., Wallace AJ, Stillman TJ, Atkins A, Jamieson SJ, Bullough PA, Green J, Artymiuk PJ, Cell. 2000 Jan 21;100(2):265-76. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/10660049 10660049]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Artymiuk, P J.]]
[[Category: Atkins, A.]]
[[Category: Bullough, P A.]]
[[Category: Green, J.]]
[[Category: Jamieson, S J.]]
[[Category: Stillman, T J.]]
[[Category: Wallace, A J.]]
[[Category: Cytolysin]]
[[Category: Hemolysin]]
[[Category: Membrane pore former]]
[[Category: Pore]]
[[Category: Toxin]]

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:22:40 2008''

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T18:06:44Z

Luis E Ramirez-Tapia:

<Structure load='1QOY' size='500' frame='true' align='right' caption='Insert caption here' scene='<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_4/Clya/5'>TextToBeDisplayed</scene>' />

{{Seed}}
[[Image:1qoy.png|left|200px]]


{{STRUCTURE_1qoy| PDB=1qoy | SCENE= }}

===E.COLI HEMOLYSIN E (HLYE, CLYA, SHEA)===


{{ABSTRACT_PUBMED_10660049}}

==About this Structure==
1QOY is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOY OCA].

==Reference==
E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy., Wallace AJ, Stillman TJ, Atkins A, Jamieson SJ, Bullough PA, Green J, Artymiuk PJ, Cell. 2000 Jan 21;100(2):265-76. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/10660049 10660049]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Artymiuk, P J.]]
[[Category: Atkins, A.]]
[[Category: Bullough, P A.]]
[[Category: Green, J.]]
[[Category: Jamieson, S J.]]
[[Category: Stillman, T J.]]
[[Category: Wallace, A J.]]
[[Category: Cytolysin]]
[[Category: Hemolysin]]
[[Category: Membrane pore former]]
[[Category: Pore]]
[[Category: Toxin]]

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:22:40 2008''

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T17:39:33Z

Luis E Ramirez-Tapia:

{{Seed}}
[[Image:1qoy.png|left|200px]]


{{STRUCTURE_1qoy| PDB=1qoy | SCENE= }}

===E.COLI HEMOLYSIN E (HLYE, CLYA, SHEA)===


{{ABSTRACT_PUBMED_10660049}}

==About this Structure==
1QOY is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOY OCA].

==Reference==
E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy., Wallace AJ, Stillman TJ, Atkins A, Jamieson SJ, Bullough PA, Green J, Artymiuk PJ, Cell. 2000 Jan 21;100(2):265-76. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/10660049 10660049]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Artymiuk, P J.]]
[[Category: Atkins, A.]]
[[Category: Bullough, P A.]]
[[Category: Green, J.]]
[[Category: Jamieson, S J.]]
[[Category: Stillman, T J.]]
[[Category: Wallace, A J.]]
[[Category: Cytolysin]]
[[Category: Hemolysin]]
[[Category: Membrane pore former]]
[[Category: Pore]]
[[Category: Toxin]]

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:22:40 2008''

User:Luis E Ramirez-Tapia/Sandbox 4

2011-12-07T17:38:03Z

Luis E Ramirez-Tapia: New page: <applet load='1ema' size='450' frame='true' align='right' scene='Green_Fluorescent_Protein/1ema_gfp_default/2' caption='Green fluorescent protein complex with peptide-derived chromophore (...

<applet load='1ema' size='450' frame='true' align='right' scene='Green_Fluorescent_Protein/1ema_gfp_default/2' caption='Green fluorescent protein complex with peptide-derived chromophore ([[1ema]])' />
'''Green fluorescent protein (GFP)''' is a [[bioluminescent]] polypeptide consisting of 238 residues isolated from the body of [[Aequorea victoria]] jellyfish.<ref name="PDBsum">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=main.html], Protein Database (PDBsum): 1ema. European Bioinformatics (EBI); 2009.</ref> GFP converts the blue chemiluminescent of [[aequorin]] in the jellyfish into green fluorescent light.<ref name="Yang">[http://www-bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html], Yang F, Moss LG, Phillips GN Jr. 1996. The molecular structure of green fluorescent protein. Biotechnology. 14: 1246-1251. DOI 10.1038/nbt1096-1246.</ref> It remains unclear why these jellyfish use fluorescence, why green is better than blue, or why they produce a separate protein for green fluorescence as opposed to simply mutating the present aequorin to shift its wavelength,<ref name="Tsien" /> but in the laboratory, GFP can be incorporated into a variety of biological systems in order to function as a marker protein. Since its discovery in 1962, GFP has become a significant contributor to the research of monitoring gene expression, localization, mobility, traffic, interactions between various membrane and cytoplasmic proteins, as well as many others.<ref name="Haldar">[http://www.springerlink.com/content/wvg513864266g77n/fulltext.pdf], Haldar S, Chattopadhyay A. 2009. The green journey. J Fluoresc. 19:1-2. DOI 10.1007/s10895-008-0455-6; biographical background on [http://en.wikipedia.org/wiki/Douglas_Prasher Douglas Prasher], [http://en.wikipedia.org/wiki/Martin_Chalfie Martin Chalfie] and [http://en.wikipedia.org/wiki/Roger_Tsien Roger Tsien].</ref>

Molecular Playground/T7 RNAP Conformations

2011-05-11T14:15:19Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'/>
</td></tr>
<tr><td>
Color code:
 
N-Terminus Domain,
Subdomain H,
Helices C1 and C2,
Specificity Loop,
Non-template Strand,
Template Strand and the
Nascent RNA Strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

Molecular Playground/T7 RNAP Conformations

2011-05-11T14:12:06Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'/>
</td></tr>
<tr><td>
Color code:
 
N-Terminus Domain,
Subdomain H,
Helices C1 and C2,
Specificity Loop,
Non-template Strand,
Template Strand and the
Nascent RNA Strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

CBI Molecules

2011-05-11T14:07:40Z

Luis E Ramirez-Tapia:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground]. Follow the links below to read nontechnical descriptions in Proteopedia.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

It's great to build on a previous entry, but you must leave the earlier one intact and make a copy that you improve and mark as new. Later we will retain the new one with a merged author list.

Fall 2010: New entries are due 12/10/10. Please append the designation (new Fall 2010) to your new entries.

Spring 2011: New entries are due 5/4/11. Please append the designation (new Spring 2011) to your new entries

Bhatia Lab
: '''[[Molecular Playground/Alginate]]''', David Griffin

: '''[[Molecular Playground/Alginate-Fall2010]]''', Joe White, David Griffin (new Fall 2010)

: '''[[Molecular Playground/Poly(ethylene glycol)]]''', Erika M. Saffer (new Spring 2011)

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau (new Fall 2010)

Forbes Lab

: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

: '''[[Molecular Playground/CheR]]''', Miaomin Zhang (new Spring 2011)

: '''[[Molecular Playground/RBP]]''', Jan Panteli (new Spring 2011)

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon

: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy
:: Best Overall CBI Molecule 2010

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Caspase-7 Dynamics]]'''

: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber

: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez

: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls

: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

: '''[[Molecular Playground/Saposin C]]''', Abla Tannous (New Spring 2011)

[http://people.biochem.umass.edu/aheuck/aph.html Heuck Lab]

: '''[[Molecular_Playground/Pcr H]]''', Fabian Romano (New Fall 2011)

Kaltashov Lab

: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita

Knapp lab

:'''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin
:: Best CBI Molecule Proteopedia Page 2010

: '''[[Molecular Playground/FIH]]''', Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/T7 RNAP Conformations]]''', Luis E. Ramirez-Tapia (New Spring 2011)

[http://robertsgroup.ecs.umass.edu/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Rohan Patil, Sarah Wilson

: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino

: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang

: '''[[Molecular playground/Chymotrypsin]]''', Brad

: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

Schnarr Lab

: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso

: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad

Thayumanavan Lab

: '''[[Molecular Playground/Lysozime ]]''', Daniella Gonzalez

: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura

: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi

: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy

: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado

: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang

: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo

: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson] & [http://www.chem.umass.edu/~rmweis/weislab/ Weis] Labs

: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto
:: Best CBI Molecule Jmol scenes 2010

: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou

:'''[[Molecular Playground/ Copper-Zinc Superoxide Dismutase]]''', Shaynah Browne (NEW FALL 2010)

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

Instructions:

'''Congratulations to the prize-winning CBI molecules noted above! These also provide great examples to follow. Another prize for best CBI Molecules page and/or scene will be awarded in spring 2011!'''

Choose a molecule that is part of your research project.

1. If you don't already have one, request a Proteopedia account and log in. If you are new to Proteopedia, click Help in the navigation box on the left to get started.

2. Make yourself a sandbox page in which you will develop your CBI Molecule scene and description (Enter "User:your name/sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. See example [[User:Lynmarie K Thompson/Sandbox 1]]).

3. Follow the format of the sample CBI molecule page [[Molecular Playground/Bacterial Chemotaxis Receptors]]. Easiest way to do this is to copy this page (in editing mode), paste it into your sandbox page, keep the first paragraph about CBI molecules, and then edit to describe and display your molecule. Your goal is to make this an interesting, nontechnical description of the molecule. If multiple people in one group work on the same molecule, you can each make different scenes for the same CBI molecule and each describe them on the same proteopedia page. Talk with each other about your plans so you are not duplicating efforts.

4. Create an attractive scene for your molecule: use the scene authoring tools in the edit mode to create the view you like, then copy the wiki text into your window.

5. Choose a "green scene" on your Proteopedia page to suggest for display at the Molecular Playground and specify a "banner" which will be projected with the molecule. This should be a short, one-line headline for your scene that includes the name of the molecule and what is important about the scene or the molecule. Remember to design this for the general public, including non-scientists. My example is: "Molecular Playground banner: A bacterial chemotaxis receptor protein used by bacteria to "smell" their environment."

6. When you are happy with your sandbox page, make yourself a permanent Proteopedia page, which will be editable by others. Enter "Molecular Playground/your molecule" (omit quotes) in the search box, then follow the instructions to create a new page with this title. Copy the content of your sandbox to this new page.

7. When you have finished the final version of your page and scene, edit this CBI Molecules page to add a listing and link for your molecule, following the "Bacterial chemotaxis receptors" example above. Please list your lab group, with your name in parentheses. That way more than one name can be associated with a Molecular Playground page (if there are several students on the Molecular Playground page, please indicate your scene with your initials). It would be great to link the lab names to web pages too.

CBI Molecules

2011-05-03T22:48:28Z

Luis E Ramirez-Tapia:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground]. Follow the links below to read nontechnical descriptions in Proteopedia.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

It's great to build on a previous entry, but you must leave the earlier one intact and make a copy that you improve and mark as new. Later we will retain the new one with a merged author list.

Fall 2010: New entries are due 12/10/10. Please append the designation (new Fall 2010) to your new entries.

Spring 2011: New entries are due 5/4/11. Please append the designation (new Spring 2011) to your new entries

Bhatia Lab
: '''[[Molecular Playground/Alginate]]''', David Griffin

: '''[[Molecular Playground/Alginate-Fall2010]]''', Joe White, David Griffin (new Fall 2010)

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau (new Fall 2010)

Forbes Lab

: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

: '''[[Molecular Playground/CheR]]''', Miaomin Zhang (new Spring 2011)

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon

: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy
:: Best Overall CBI Molecule 2010

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Caspase-7 Dynamics]]'''

: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber

: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez

: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls

: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

[http://people.biochem.umass.edu/aheuck/aph.html Heuck Lab]

: '''[[Molecular_Playground/Pcr H]]''', Fabian Romano (new Fall 2011)

Kaltashov Lab

: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita

Knapp lab

:'''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin
:: Best CBI Molecule Proteopedia Page 2010

: '''[[Molecular Playground/FIH]]''', Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/T7 RNAP Conformations]]''', Luis E. Ramirez-Tapia (new Fall 2010)

[http://robertsgroup.ecs.umass.edu/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Rohan Patil, Sarah Wilson

: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino

: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang

: '''[[Molecular playground/Chymotrypsin]]''', Brad

: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

Schnarr Lab

: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso

: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad

Thayumanavan Lab

: '''[[Molecular Playground/Lysozime ]]''', Daniella Gonzalez

: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura

: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi

: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy

: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado

: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang

: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo

: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson] & [http://www.chem.umass.edu/~rmweis/weislab/ Weis] Labs

: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto
:: Best CBI Molecule Jmol scenes 2010

: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou

:'''[[Molecular Playground/ Copper-Zinc Superoxide Dismutase]]''', Shaynah Browne

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

Instructions:

'''Congratulations to the prize-winning CBI molecules noted above! These also provide great examples to follow. Another prize for best CBI Molecules page and/or scene will be awarded in spring 2011!'''

Choose a molecule that is part of your research project.

1. If you don't already have one, request a Proteopedia account and log in. If you are new to Proteopedia, click Help in the navigation box on the left to get started.

2. Make yourself a sandbox page in which you will develop your CBI Molecule scene and description (Enter "User:your name/sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. See example [[User:Lynmarie K Thompson/Sandbox 1]]).

3. Follow the format of the sample CBI molecule page [[Molecular Playground/Bacterial Chemotaxis Receptors]]. Easiest way to do this is to copy this page (in editing mode), paste it into your sandbox page, keep the first paragraph about CBI molecules, and then edit to describe and display your molecule. Your goal is to make this an interesting, nontechnical description of the molecule. If multiple people in one group work on the same molecule, you can each make different scenes for the same CBI molecule and each describe them on the same proteopedia page. Talk with each other about your plans so you are not duplicating efforts.

4. Create an attractive scene for your molecule: use the scene authoring tools in the edit mode to create the view you like, then copy the wiki text into your window.

5. Choose a "green scene" on your Proteopedia page to suggest for display at the Molecular Playground and specify a "banner" which will be projected with the molecule. This should be a short, one-line headline for your scene that includes the name of the molecule and what is important about the scene or the molecule. Remember to design this for the general public, including non-scientists. My example is: "Molecular Playground banner: A bacterial chemotaxis receptor protein used by bacteria to "smell" their environment."

6. When you are happy with your sandbox page, make yourself a permanent Proteopedia page, which will be editable by others. Enter "Molecular Playground/your molecule" (omit quotes) in the search box, then follow the instructions to create a new page with this title. Copy the content of your sandbox to this new page.

7. When you have finished the final version of your page and scene, edit this CBI Molecules page to add a listing and link for your molecule, following the "Bacterial chemotaxis receptors" example above. Please list your lab group, with your name in parentheses. That way more than one name can be associated with a Molecular Playground page (if there are several students on the Molecular Playground page, please indicate your scene with your initials). It would be great to link the lab names to web pages too.

CBI Molecules

2011-05-02T15:37:48Z

Luis E Ramirez-Tapia:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground]. Follow the links below to read nontechnical descriptions in Proteopedia.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

It's great to build on a previous entry, but you must leave the earlier one intact and make a copy that you improve and mark as new. Later we will retain the new one with a merged author list.

Fall 2010: New entries are due 12/10/10. Please append the designation (new Fall 2010) to your new entries.

Spring 2011: New entries are due 5/4/11. Please append the designation (new Spring 2011) to your new entries

Bhatia Lab
: '''[[Molecular Playground/Alginate]]''', David Griffin

: '''[[Molecular Playground/Alginate-Fall2010]]''', Joe White, David Griffin (new Fall 2010)

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau (new Fall 2010)

Forbes Lab

: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon

: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy
:: Best Overall CBI Molecule 2010

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Caspase-7 Dynamics]]'''

: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber

: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez

: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls

: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

[http://people.biochem.umass.edu/aheuck/aph.html Heuck Lab]

: '''[[Molecular_Playground/Pcr H]]''', Fabian Romano

Kaltashov Lab

: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita

Knapp lab

:'''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin
:: Best CBI Molecule Proteopedia Page 2010

: '''[[Molecular Playground/FIH]]''', Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/T7 RNAP Conformations]]''', Luis E. Ramirez-Tapia (new Fall 2010)

[http://robertsgroup.ecs.umass.edu/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Rohan Patil, Sarah Wilson

: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino

: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang

: '''[[Molecular playground/Chymotrypsin]]''', Brad

: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

Schnarr Lab

: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso

: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad

Thayumanavan Lab

: '''[[Molecular Playground/Lysozime ]]''', Daniella Gonzalez

: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura

: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi

: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy

: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado

: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang

: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo

: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson] & [http://www.chem.umass.edu/~rmweis/weislab/ Weis] Labs

: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto
:: Best CBI Molecule Jmol scenes 2010

: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

Instructions:

'''Congratulations to the prize-winning CBI molecules noted above! These also provide great examples to follow. Another prize for best CBI Molecules page and/or scene will be awarded in spring 2011!'''

Choose a molecule that is part of your research project.

1. If you don't already have one, request a Proteopedia account and log in. If you are new to Proteopedia, click Help in the navigation box on the left to get started.

2. Make yourself a sandbox page in which you will develop your CBI Molecule scene and description (Enter "User:your name/sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. See example [[User:Lynmarie K Thompson/Sandbox 1]]).

3. Follow the format of the sample CBI molecule page [[Molecular Playground/Bacterial Chemotaxis Receptors]]. Easiest way to do this is to copy this page (in editing mode), paste it into your sandbox page, keep the first paragraph about CBI molecules, and then edit to describe and display your molecule. Your goal is to make this an interesting, nontechnical description of the molecule. If multiple people in one group work on the same molecule, you can each make different scenes for the same CBI molecule and each describe them on the same proteopedia page. Talk with each other about your plans so you are not duplicating efforts.

4. Create an attractive scene for your molecule: use the scene authoring tools in the edit mode to create the view you like, then copy the wiki text into your window.

5. Choose a "green scene" on your Proteopedia page to suggest for display at the Molecular Playground and specify a "banner" which will be projected with the molecule. This should be a short, one-line headline for your scene that includes the name of the molecule and what is important about the scene or the molecule. Remember to design this for the general public, including non-scientists. My example is: "Molecular Playground banner: A bacterial chemotaxis receptor protein used by bacteria to "smell" their environment."

6. When you are happy with your sandbox page, make yourself a permanent Proteopedia page, which will be editable by others. Enter "Molecular Playground/your molecule" (omit quotes) in the search box, then follow the instructions to create a new page with this title. Copy the content of your sandbox to this new page.

7. When you have finished the final version of your page and scene, edit this CBI Molecules page to add a listing and link for your molecule, following the "Bacterial chemotaxis receptors" example above. Please list your lab group, with your name in parentheses. That way more than one name can be associated with a Molecular Playground page (if there are several students on the Molecular Playground page, please indicate your scene with your initials). It would be great to link the lab names to web pages too.

Molecular Playground/Pcr H

2011-05-02T15:37:11Z

Luis E Ramirez-Tapia: New page: One of the CBI Molecules being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at th...

CBI Molecules

2011-04-29T05:59:32Z

Luis E Ramirez-Tapia:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground]. Follow the links below to read nontechnical descriptions in Proteopedia.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

It's great to build on a previous entry, but you must leave the earlier one intact and make a copy that you improve and mark as new. Later we will retain the new one with a merged author list.

Fall 2010: New entries are due 12/10/10. Please append the designation (new Fall 2010) to your new entries.

Spring 2011: New entries are due 5/4/11. Please append the designation (new Spring 2011) to your new entries

Bhatia Lab
: '''[[Molecular Playground/Alginate]]''', David Griffin

: '''[[Molecular Playground/Alginate-Fall2010]]''', Joe White, David Griffin (new Fall 2010)

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau (new Fall 2010)

Forbes Lab

: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon

: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy
:: Best Overall CBI Molecule 2010

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Caspase-7 Dynamics]]'''

: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber

: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez

: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls

: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

Kaltashov Lab

: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita

Knapp lab

:'''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin
:: Best CBI Molecule Proteopedia Page 2010

: '''[[Molecular Playground/FIH]]''', Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/T7 RNAP Conformations]]''', Luis E. Ramirez-Tapia (new Fall 2010)

[http://robertsgroup.ecs.umass.edu/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Rohan Patil, Sarah Wilson

: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino

: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang

: '''[[Molecular playground/Chymotrypsin]]''', Brad

: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

Schnarr Lab

: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso

: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad

Thayumanavan Lab

: '''[[Molecular Playground/Lysozime ]]''', Daniella Gonzalez

: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura

: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi

: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy

: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado

: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang

: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo

: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson] & [http://www.chem.umass.edu/~rmweis/weislab/ Weis] Labs

: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto
:: Best CBI Molecule Jmol scenes 2010

: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

Instructions:

'''Congratulations to the prize-winning CBI molecules noted above! These also provide great examples to follow. Another prize for best CBI Molecules page and/or scene will be awarded in spring 2011!'''

Choose a molecule that is part of your research project.

1. If you don't already have one, request a Proteopedia account and log in. If you are new to Proteopedia, click Help in the navigation box on the left to get started.

2. Make yourself a sandbox page in which you will develop your CBI Molecule scene and description (Enter "User:your name/sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. See example [[User:Lynmarie K Thompson/Sandbox 1]]).

3. Follow the format of the sample CBI molecule page [[Molecular Playground/Bacterial Chemotaxis Receptors]]. Easiest way to do this is to copy this page (in editing mode), paste it into your sandbox page, keep the first paragraph about CBI molecules, and then edit to describe and display your molecule. Your goal is to make this an interesting, nontechnical description of the molecule. If multiple people in one group work on the same molecule, you can each make different scenes for the same CBI molecule and each describe them on the same proteopedia page. Talk with each other about your plans so you are not duplicating efforts.

4. Create an attractive scene for your molecule: use the scene authoring tools in the edit mode to create the view you like, then copy the wiki text into your window.

5. Choose a "green scene" on your Proteopedia page to suggest for display at the Molecular Playground and specify a "banner" which will be projected with the molecule. This should be a short, one-line headline for your scene that includes the name of the molecule and what is important about the scene or the molecule. Remember to design this for the general public, including non-scientists. My example is: "Molecular Playground banner: A bacterial chemotaxis receptor protein used by bacteria to "smell" their environment."

6. When you are happy with your sandbox page, make yourself a permanent Proteopedia page, which will be editable by others. Enter "Molecular Playground/your molecule" (omit quotes) in the search box, then follow the instructions to create a new page with this title. Copy the content of your sandbox to this new page.

7. When you have finished the final version of your page and scene, edit this CBI Molecules page to add a listing and link for your molecule, following the "Bacterial chemotaxis receptors" example above. Please list your lab group, with your name in parentheses. That way more than one name can be associated with a Molecular Playground page (if there are several students on the Molecular Playground page, please indicate your scene with your initials). It would be great to link the lab names to web pages too.

CBI Molecules

2011-04-29T05:58:08Z

Luis E Ramirez-Tapia:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground]. Follow the links below to read nontechnical descriptions in Proteopedia.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

It's great to build on a previous entry, but you must leave the earlier one intact and make a copy that you improve and mark as new. Later we will retain the new one with a merged author list.

Fall 2010: New entries are due 12/10/10. Please append the designation (new Fall 2010) to your new entries.

Spring 2011: New entries are due 5/4/11. Please append the designation (new Spring 2011) to your new entries

Bhatia Lab
: '''[[Molecular Playground/Alginate]]''', David Griffin

: '''[[Molecular Playground/Alginate-Fall2010]]''', Joe White, David Griffin (new Fall 2010)

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau (new Fall 2010)

Forbes Lab

: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon

: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy
:: Best Overall CBI Molecule 2010

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Caspase-7 Dynamics]]'''

: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber

: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez

: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls

: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

Kaltashov Lab

: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita

Knapp lab

:'''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin
:: Best CBI Molecule Proteopedia Page 2010

: '''[[Molecular Playground/FIH]]''', Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/T7 RNAP Comformations]]''', Luis E. Ramirez-Tapia (new Fall 2010)

[http://robertsgroup.ecs.umass.edu/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Rohan Patil, Sarah Wilson

: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino

: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang

: '''[[Molecular playground/Chymotrypsin]]''', Brad

: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

Schnarr Lab

: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso

: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad

Thayumanavan Lab

: '''[[Molecular Playground/Lysozime ]]''', Daniella Gonzalez

: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura

: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi

: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy

: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado

: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang

: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo

: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson] & [http://www.chem.umass.edu/~rmweis/weislab/ Weis] Labs

: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto
:: Best CBI Molecule Jmol scenes 2010

: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

Instructions:

'''Congratulations to the prize-winning CBI molecules noted above! These also provide great examples to follow. Another prize for best CBI Molecules page and/or scene will be awarded in spring 2011!'''

Choose a molecule that is part of your research project.

1. If you don't already have one, request a Proteopedia account and log in. If you are new to Proteopedia, click Help in the navigation box on the left to get started.

2. Make yourself a sandbox page in which you will develop your CBI Molecule scene and description (Enter "User:your name/sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. See example [[User:Lynmarie K Thompson/Sandbox 1]]).

3. Follow the format of the sample CBI molecule page [[Molecular Playground/Bacterial Chemotaxis Receptors]]. Easiest way to do this is to copy this page (in editing mode), paste it into your sandbox page, keep the first paragraph about CBI molecules, and then edit to describe and display your molecule. Your goal is to make this an interesting, nontechnical description of the molecule. If multiple people in one group work on the same molecule, you can each make different scenes for the same CBI molecule and each describe them on the same proteopedia page. Talk with each other about your plans so you are not duplicating efforts.

4. Create an attractive scene for your molecule: use the scene authoring tools in the edit mode to create the view you like, then copy the wiki text into your window.

5. Choose a "green scene" on your Proteopedia page to suggest for display at the Molecular Playground and specify a "banner" which will be projected with the molecule. This should be a short, one-line headline for your scene that includes the name of the molecule and what is important about the scene or the molecule. Remember to design this for the general public, including non-scientists. My example is: "Molecular Playground banner: A bacterial chemotaxis receptor protein used by bacteria to "smell" their environment."

6. When you are happy with your sandbox page, make yourself a permanent Proteopedia page, which will be editable by others. Enter "Molecular Playground/your molecule" (omit quotes) in the search box, then follow the instructions to create a new page with this title. Copy the content of your sandbox to this new page.

7. When you have finished the final version of your page and scene, edit this CBI Molecules page to add a listing and link for your molecule, following the "Bacterial chemotaxis receptors" example above. Please list your lab group, with your name in parentheses. That way more than one name can be associated with a Molecular Playground page (if there are several students on the Molecular Playground page, please indicate your scene with your initials). It would be great to link the lab names to web pages too.

Molecular Playground/T7 RNAP Conformations

2011-04-29T05:55:18Z

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code:
 
N-Terminus Domain,
Subdomain H,
Helices C1 and C2,
Specificity Loop,
Non-template Strand,
Template Strand and the
Nascent RNA Strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:21:22Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code:
 
N-Terminus Domain,
Subdomain H,
Helices C1 and C2,
Specificity Loop,
Non-template Strand,
Template Strand and the
Nascent RNA Strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:20:15Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code:
 
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:19:02Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:14:55Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: initiation state (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation state (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:11:55Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were feeded are. initiation state (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation state (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:04:25Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:02:52Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T05:00:44Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:59:47Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:51:03Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:45:33Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach could require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:44:26Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:41:27Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
 
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:40:36Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:38:29Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>the most notorious conformational change</scene> shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it? Follow the movement of the green helices. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:33:35Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the movement of the green helices. In deed, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:32:51Z

Luis E Ramirez-Tapia: /* Understanding the Morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the movement of the green helices. In deed, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:29:52Z

Luis E Ramirez-Tapia: /* Understanding the morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the Morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the following button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the movement of the green helices. In deed, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:26:59Z

Luis E Ramirez-Tapia: /* Understanding the morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, press the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the movement of the green helices. In deed, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:24:41Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.
----
===Understanding the morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex press the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:23:28Z

Luis E Ramirez-Tapia: /* Understanding the morph */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex press the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:22:08Z

Luis E Ramirez-Tapia:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:20:03Z

Luis E Ramirez-Tapia:

<body align="justify">
One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</body>

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:12:46Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|400px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:12:12Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|350px|left| Figure 1. Abortive Cycle during transcription initiation]]

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:11:45Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png|thumb|350px|left| Figure 1. Abortive Cycle during transcription initiation

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:07:23Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png]]
 Figure 1. Abortive Cycle during transcription initiation 

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]

User:Luis E Ramirez-Tapia/T7 RNA polymerase

2011-04-29T04:04:03Z

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground]
----
<table align="right" width="300" border="0" style="background-color:#e0e0e0;"><tr><td>
<Structure load='1qln' size='400' frame ='true' align ='right' caption='T7 RNA polymerase' scene='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'/>
</td></tr>
<tr><td>
Color code
N-Terminus domain,
Subdomain H,
Helices C1 and C2,
specificity loop,
Non-template strand,
template strand and the
nascent RNA strand
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>
*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/2'>Elongation state = 17 mer scaffold </scene>
</td></tr></table>

= Conformational Changes on T7 RNA Polymerase =

[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood.
 
[[Image:Abortivecycling.png]]
 Figure 1. Abortive Cycle during transcription initiation 

In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/proline266/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focus on resolving the mechanism behind this mutation.

===Understanding the morph===
You can see the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex by pressing the follow button.
<jmol>
<jmolButton>
<script>
script "/wiki/images/5/51/Rnaptransition.spt"
</script>
<text>Play Animation</text>
</jmolButton>
</jmol>
The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2.
The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'>HUGE CONFORMATIONAL CHANGE</scene>, shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances and make a model of the correct transition. That is work in progress...
Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).

=References=

#Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
#Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.

=Acknowledgement=

To Professor Eric Martz his advice was crucial to develop this page.

=See Also=
*[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]
*[http://www.youtube.com/watch?v=veY0LlL7Dt0 PcrA Helicase Morph] [http://www.proteopedia.org/wiki/index.php/User:Luis_E_Ramirez-Tapia/Sandbox_2 PcrA Helicase]