T7 RNA Polymerase: Difference between revisions

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==T7 RNA Polymerase Initiation Complex==
==T7 RNA Polymerase Initiation Complex==
<StructureSection load='1qln' size='450' side='right' caption='Promoter bound polymerase' scene=''>
<StructureSection load='1qln' size='450' side='right' caption='Promoter bound polymerase (PDB code [[1qln]])' scene=''>
'''Promoter binding'''. T7 RNA polymerase <scene name='77/778917/T7rp_promo_bound_basic/1'>binds and melts into dsDNA</scene>, by recognizing the upstream duplex region of the promoter (-17 to -5), and then melting a bubble (-4 to about +3), within the larger duplex. The duplex promoter domain binds primarily to the N-terminal domain of the enzyme, with the exception of the (C-terminal domain) "<scene name='77/778917/T7rp_promo_bound_specif_loop_z/2'>specificity loop</scene>." It is the combination of the N-terminal domain, with the positioned specificity loop, that forms the specific binding surface.
'''Promoter binding'''. T7 RNA polymerase <scene name='77/778917/T7rp_promo_bound_basic/1'>binds and melts into dsDNA</scene>, by recognizing the <scene name='77/778917/T7rp_promo_highlight_aa/1'>upstream duplex region</scene> of the promoter (-17 to -5), and then melting a bubble (-4 to about +3), within the larger duplex. The duplex promoter domain binds primarily to the N-terminal domain of the enzyme, with the exception of the (C-terminal domain) "<scene name='77/778917/T7rp_promo_bound_specif_loop_z/2'>specificity loop</scene>." It is the combination of the N-terminal domain, with the positioned specificity loop, that forms the specific binding surface.


'''Intercalating loop stabilizes the melted complex'''. Strong interactions with the duplex region of the promoter places the "<scene name='77/778917/T7rp_promo_bound_val_loop_z/1'>intercalating loop</scene>" into the DNA between residues -4 and -5. The intercalating loop, also called the Valine Loop, has hydrophobic residues Val, Ile, etc that stack on and stabilize the exposed face of the base pair at position -5, stabilizing the locally melted structure.
'''Intercalating loop stabilizes the melted complex'''. Strong interactions with the duplex region of the promoter places the "<scene name='77/778917/T7rp_promo_bound_val_loop_z/1'>intercalating loop</scene>" into the DNA between residues -4 and -5. The intercalating loop, also called the Valine Loop, has hydrophobic residues Val, Ile, etc that stack on and stabilize the exposed face of the base pair at position -5, stabilizing the locally melted structure.
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'''The initial bubble grows'''. From 2mer, the system progresses ->3mer->4mer->5mer->6mer ...  During this time, the newly formed RNA-DNA duplex (hybrid) grows, from 2 bases, to <scene name='77/778917/T7rp_3mer_rna_backedout/1'>3 bases</scene>, to 4 bases, to 5 bases, to 6 bases, to<scene name='77/778917/T7rp_7mer_rna_backedout/1'> 7 bases</scene>, to <scene name='77/778917/T7rp_8mer_rna_backedout/1'>8 bases</scene>, etc., and during the time, the duplex is short and otherwise unstable.  
'''The initial bubble grows'''. From 2mer, the system progresses ->3mer->4mer->5mer->6mer ...  During this time, the newly formed RNA-DNA duplex (hybrid) grows, from 2 bases, to <scene name='77/778917/T7rp_3mer_rna_backedout/1'>3 bases</scene>, to 4 bases, to 5 bases, to 6 bases, to<scene name='77/778917/T7rp_7mer_rna_backedout/1'> 7 bases</scene>, to <scene name='77/778917/T7rp_8mer_rna_backedout/1'>8 bases</scene>, etc., and during the time, the duplex is short and otherwise unstable.  


'''The growing hybrid induces protein domain movement'''. Also note that the initial active site accommodates only about a 3 base RNA-DNA duplex, as the N-terminal domain lies in the path of that hybrid (remember that forward translocation of the polymerase is really reverse translocation of the RNA-DNA hybrid). Beyond about 3 bases, the hybrid pushes on the N-terminal domain, inducing into to both translate backwards and rotate. This can be seen in structures of the complex with 7 and 8 bases of RNA synthesized.
'''The growing hybrid induces protein domain movement'''. Also note that the initial active site accommodates only about a 3 basepair RNA-DNA duplex, as the N-terminal domain lies in the path of that hybrid (remember that forward translocation of the polymerase is really reverse translocation of the RNA-DNA hybrid). Beyond about 3 bases, the <scene name='77/778917/Morph/1'>hybrid pushes on the N-terminal domain</scene>, inducing into to both translate backwards and rotate. This can be seen in structures of the complex with 7 and 8 nucleotides of RNA synthesized. In the morph shown <ref>The [[Jmol/Storymorph|Storymorph Jmol scripts]] were used to create the interpolation shown in the morph. [https://proteopedia.org/wiki/index.php/Image:Morph_timing_t7rnap.pdb Coordinates] available on Proteopedia</ref>, models of how the structure might look with 4, 5, and 6 nucleotides of RNA synthesized were interpolated from the existing structures with 3 and 7 nucleotides of RNA. In this <jmol>
  <jmolLink>
    <script> anim off; delay 0.5; model 0; display 1.1; delay 0.5;
display 1.2 or 1.1 and (RNA or 14); delay 0.5
display 1.3 or 1.1 and (RNA or 14-15); delay 0.5
display 1.4 or 1.1 and (RNA or 14-16); delay 0.5
display 1.5 or 1.1 and (RNA or 14-16) or 1.2 and RNA; delay 1.5;display all;model 1;
</script>
    <text>alternate sequence</text>
  </jmolLink>
</jmol>, the growing hybrid is shown explicitly (with some modeling problem for 6 nucleotides if you look closely).
 
'''Transition to Elongation'''. Toward the end of the above rotation (at about a 9mer RNA), stress builds up in the promoter binding domain, leading to a weakening of some of the interactions with the upstream duplex promoter. This triggers promoter release, which now allows the N-terminal domain to rotate 220° in the ''other'' direction, to form the <scene name='77/778917/T7rp_elongation_cmplx_full_vw/1'>elongation complex</scene>.


== Function ==
== Function ==
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</StructureSection>
</StructureSection>
== Files for 3D printer ==
<i class="fas fa-cubes"></i> T7 RNA polymerase model by [[User:Marius Mihasan|Marius Mihasan]] [https://3dprint.nih.gov/discover/3dpx-015377  <i class="fas fa-download"></i>]
== References ==
== References ==
1,"1aro","Free enz plus lysozyme","T7RP with the bound inhibitor T7 lysozyme, no DNA - Jeruzalmi, D. & Steitz, T. A. (1998) EMBO J 17, 4101-4113"
1,"1aro","Free enz plus lysozyme","T7RP with the bound inhibitor T7 lysozyme, no DNA - Jeruzalmi, D. & Steitz, T. A. (1998) EMBO J 17, 4101-4113"
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<references/>
<references/>
[[Category:3D printer files]]
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