User:Nathan Harris/Tus: Difference between revisions

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Tus is divided into an <scene name='User:Nathan_Harris/Tus/Amino_domian/1'>amino domain</scene> and <scene name='User:Nathan_Harris/Tus/Carboxy_domain/1'>carboxy domain</scene> distinguished by two alpha helical regions and central β sheets combining to encompass a large central basic cleft. The <scene name='User:Nathan_Harris/Tus/Interdomain/2'>interdomain region</scene> consists of anti-parallel β strands and an <scene name='User:Nathan_Harris/Tus/L4/1'>extended L4 loop</scene> which connect the amino and carboxy domains. Within this interdomain region, the <scene name='User:Nathan_Harris/Tus/Bf/1'>βF</scene>, <scene name='User:Nathan_Harris/Tus/Bg/1'>βG</scene>, <scene name='User:Nathan_Harris/Tus/Bh/1'>βH</scene> and <scene name='User:Nathan_Harris/Tus/Bi/1'>βI</scene> strands are responsible for specific and non-specific recognition of ''Ter''.

The amino domain consists of three amphipathic alpha helices forming an anti-parallel bundle roughly parallel to ''Ter'', a sandwich of anti-parallel β sheets and three loops. The major groove and minor groove are clamped by two alpha helices (<scene name='User:Nathan_Harris/Tus/A4/1'>αIV</scene> and <scene name='User:Nathan_Harris/Tus/A5/1'>αV</scene>) which also contribute to the hydrophobic core of the protein. Within the β sandwich, <scene name='User:Nathan_Harris/Tus/Bcadke/1'>βCADKE</scene> contacts the alpha helical region, whereas <scene name='User:Nathan_Harris/Tus/Blfij/1'>βLFIJ</scene> is associated with DNA binding. Furthermore, the extended L4 loop is also involved in contacts to the minor groove.

The carboxy domain consists of a hydrophobic core stabilised by alpha helices and β strands (βGHNO). The <scene name='User:Nathan_Harris/Tus/L3/1'>L3 loop</scene> is responsible for connecting helices <scene name='User:Nathan_Harris/Tus/A6/1'>αVI</scene> and <scene name='User:Nathan_Harris/Tus/A7/1'>αVII</scene> and also contacts the minor groove of DNA <ref name = "~~Kamada~~" /> <ref name = "~~Neylon~~" />.

The carboxy domain consists of a hydrophobic core stabilised by alpha helices and β strands (βGHNO). The <scene name='User:Nathan_Harris/Tus/L3/1'>L3 loop</scene> is responsible for connecting helices <scene name='User:Nathan_Harris/Tus/A6/1'>αVI</scene> and <scene name='User:Nathan_Harris/Tus/A7/1'>αVII</scene> and also contacts the minor groove of DNA <ref name = "Neylon" /><ref name = "Kamada" />.

=='''Confirmation changes induced on Ter sites'''==

The ''Ter'' region in ''E.coli'' between bases T5 and A9 is significantly underwound upon binding with Tus. This region of DNA is altered from standard B form which is attributed to straddling of ''Ter'' by interdomain β strands (βF and βG) and the L4 connecting loop of Tus. Tus interacts with ''Ter'' in a previously undescribed manner with β strands of Tus inserting almost perpendicularly into the major groove to recognise ''Ter''. Alteration of ''Ter'' is characterised by an extended major groove and a broadened minor groove generating an overall DNA bend of 20 degrees. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site <ref name = "~~Kamada~~" /> <ref name = "~~Neylon~~" />.

The ''Ter'' region in ''E.coli'' between bases T5 and A9 is significantly underwound upon binding with Tus. This region of DNA is altered from standard B form which is attributed to straddling of ''Ter'' by interdomain β strands (βF and βG) and the L4 connecting loop of Tus. Tus interacts with ''Ter'' in a previously undescribed manner with β strands of Tus inserting almost perpendicularly into the major groove to recognise ''Ter''. Alteration of ''Ter'' is characterised by an extended major groove and a broadened minor groove generating an overall DNA bend of 20 degrees. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site <ref name = "Neylon" /><ref name = "Kamada" />.

=='''Mechanism of action'''==

The ability of Tus to terminate replication in ''E. coli'' in a polar manner is believed to involve the inhibition of DnaB helicase. This is achieved either through a “locked complex” model provided by Tus-Ter interactions providing a physical block, protein-protein interactions between Tus and DnaB, or through a combination of these two effects.

The ability of Tus to terminate replication in ''E. coli'' in a polar manner is believed to involve the inhibition of DnaB helicase. This is achieved either through a “locked complex” model provided by Tus-Ter interactions providing a physical block, protein-protein interactions between Tus and DnaB, or through a combination of these two effects<ref name = "Kamada" /><ref name = "Kaplan" />.

===='''The Tus- ''Ter'' locked complex'''====

It has been suggested that the affinity of Tus for ''Ter'' may contribute to the polar arrest of replication in ''E. coli'' demonstrated by a direct

correlation between the affinity and replication termination.

correlation between the affinity and replication termination <ref name = "Mulcair"> Mulcair, M. D., Schaeffer, P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, T. M. and Dixon, N .E. (2006) A molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125: 1309-1319.</ref>.

Investigations of the affinity of Tus for partially unwound ''Ter'' DNA have provided crystal structures of Tus bound to ''Ter'' unwound at the <scene name='User:Nathan_Harris/Tus/C6/1'>C6</scene> of ''Ter''.

These crystal structures show the C6 of ''Ter'' flipped up into a <scene name='User:Nathan_Harris/Tus/Pocket/1'>hydrophobic pocket</scene> of Tus forming a so called locked complex.

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with Tus leading to dissociation of Tus and progression of the replication fork.

However, when ''E. coli'' ''Ter'' sequences are inserted into a plasmid in ''B. Subtillis'' expressing Tus, the replication fork arrest from the non-permissive

end only occurs with 0.5% efficiency compared to 45.4% efficiency in a wild type ''E. coli'' system. If only Tus-''Ter'' interactions were important in the

end only occurs with 0.5% efficiency compared to 45.4% efficiency in a wild type ''E. coli'' system<ref name = "Anderson"> Anderson, P., Griffith, A., Duggin, I. and Wake, R. (2000) Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E.coli. Molecular Microbiology, 36 (6): 1327-1335.</ref>. If only Tus-''Ter'' interactions were important in the

mediation of polar fork arrest, then the efficiency in the two systems should be similar. This highlights the importance of other factors in the

mediation of polar fork arrest.

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Anderson, P., Griffith, A., Duggin, I. and Wake, R. (2000) Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E.coli. Molecular Microbiology, 36 (6): 1327-1335.

Bastia, D., Zzaman, S., Krings, G., Saxena, M., Peng, X. and Greenberg, M. (2008) Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase. Proceedings of the National Academy of Science, USA, 105 (93): 12831-12836.

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Mulcair, M. D., Schaeffer, P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, T. M. and Dixon, N .E. (2006) A molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125: 1309-1319.

Mulugu, S., Potnis, A., Shamsuzzaman, T. J., Alexander, K. and Bastia, D. (2001) Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Science, USA, 98 (17): 9569-9574.

Neylon, C., Kralicek, A. V., Hill, T.M. and Dixon, N.E. (2005) Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex. Microbiology and Molecular Biology, 69 (3): 501-526.

User:Nathan Harris/Tus: Difference between revisions

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@@ Line 14: / Line 14: @@
 Tus is divided into an <scene name='User:Nathan_Harris/Tus/Amino_domian/1'>amino domain</scene> and <scene name='User:Nathan_Harris/Tus/Carboxy_domain/1'>carboxy domain</scene> distinguished by two alpha helical regions and central β sheets combining to encompass a large central basic cleft. The <scene name='User:Nathan_Harris/Tus/Interdomain/2'>interdomain region</scene> consists of anti-parallel β strands and an <scene name='User:Nathan_Harris/Tus/L4/1'>extended L4 loop</scene> which connect the amino and carboxy domains. Within this interdomain region, the <scene name='User:Nathan_Harris/Tus/Bf/1'>βF</scene>, <scene name='User:Nathan_Harris/Tus/Bg/1'>βG</scene>, <scene name='User:Nathan_Harris/Tus/Bh/1'>βH</scene> and <scene name='User:Nathan_Harris/Tus/Bi/1'>βI</scene> strands are responsible for specific and non-specific recognition of ''Ter''.
 The amino domain consists of three amphipathic alpha helices forming an anti-parallel bundle roughly parallel to ''Ter'', a sandwich of anti-parallel β sheets and three loops. The major groove and minor groove are clamped by two alpha helices (<scene name='User:Nathan_Harris/Tus/A4/1'>αIV</scene> and <scene name='User:Nathan_Harris/Tus/A5/1'>αV</scene>) which also contribute to the hydrophobic core of the protein. Within the β sandwich, <scene name='User:Nathan_Harris/Tus/Bcadke/1'>βCADKE</scene> contacts the alpha helical region, whereas <scene name='User:Nathan_Harris/Tus/Blfij/1'>βLFIJ</scene> is associated with DNA binding.  Furthermore, the extended L4 loop is also involved in contacts to the minor groove.
-The carboxy domain consists of a hydrophobic core stabilised by alpha helices and β strands (βGHNO). The <scene name='User:Nathan_Harris/Tus/L3/1'>L3 loop</scene> is responsible for connecting helices <scene name='User:Nathan_Harris/Tus/A6/1'>αVI</scene> and <scene name='User:Nathan_Harris/Tus/A7/1'>αVII</scene> and also contacts the minor groove of DNA <ref name = "Kamada" /> <ref name = "Neylon" />.
+The carboxy domain consists of a hydrophobic core stabilised by alpha helices and β strands (βGHNO). The <scene name='User:Nathan_Harris/Tus/L3/1'>L3 loop</scene> is responsible for connecting helices <scene name='User:Nathan_Harris/Tus/A6/1'>αVI</scene> and <scene name='User:Nathan_Harris/Tus/A7/1'>αVII</scene> and also contacts the minor groove of DNA <ref name = "Neylon" /><ref name = "Kamada" />.
 =='''Confirmation changes induced on Ter sites'''==
-The ''Ter'' region in ''E.coli'' between bases T5 and A9 is significantly underwound upon binding with Tus. This region of DNA is altered from standard B form which is attributed to straddling of ''Ter'' by interdomain β strands (βF and βG) and the L4 connecting loop of Tus. Tus interacts with ''Ter'' in a previously undescribed manner with β strands of Tus inserting almost perpendicularly into the major groove to recognise ''Ter''. Alteration of ''Ter'' is characterised by an extended major groove and a broadened minor groove generating an overall DNA bend of 20 degrees. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site <ref name = "Kamada" /> <ref name = "Neylon" />.
+The ''Ter'' region in ''E.coli'' between bases T5 and A9 is significantly underwound upon binding with Tus. This region of DNA is altered from standard B form which is attributed to straddling of ''Ter'' by interdomain β strands (βF and βG) and the L4 connecting loop of Tus. Tus interacts with ''Ter'' in a previously undescribed manner with β strands of Tus inserting almost perpendicularly into the major groove to recognise ''Ter''. Alteration of ''Ter'' is characterised by an extended major groove and a broadened minor groove generating an overall DNA bend of 20 degrees. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site <ref name = "Neylon" /><ref name = "Kamada" />.
 =='''Mechanism of action'''==
-The ability of Tus to terminate replication in ''E. coli'' in a polar manner is believed to involve the inhibition of DnaB helicase.  This is achieved either through a “locked complex” model provided by Tus-Ter interactions providing a physical block, protein-protein interactions between Tus and DnaB, or through a combination of these two effects.
+The ability of Tus to terminate replication in ''E. coli'' in a polar manner is believed to involve the inhibition of DnaB helicase.  This is achieved either through a “locked complex” model provided by Tus-Ter interactions providing a physical block, protein-protein interactions between Tus and DnaB, or through a combination of these two effects<ref name = "Kamada" /><ref name = "Kaplan" />.
 ===='''The Tus- ''Ter'' locked complex'''====
 It has been suggested that the affinity of Tus for ''Ter'' may contribute to the polar arrest of replication in ''E. coli'' demonstrated by a direct
-correlation between the affinity and replication termination.
+correlation between the affinity and replication termination <ref name = "Mulcair"> Mulcair, M. D., Schaeffer, P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, T. M. and Dixon, N .E.  (2006) A molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125: 1309-1319.</ref>.
 Investigations of the affinity of Tus for partially unwound ''Ter'' DNA have provided crystal structures of Tus bound to ''Ter'' unwound at the <scene name='User:Nathan_Harris/Tus/C6/1'>C6</scene> of ''Ter''.
 These crystal structures show the C6 of ''Ter'' flipped up into a <scene name='User:Nathan_Harris/Tus/Pocket/1'>hydrophobic pocket</scene> of Tus forming a so called locked complex.
@@ Line 36: / Line 35: @@
 with Tus leading to dissociation of Tus and progression of the replication fork.
 However, when ''E. coli'' ''Ter'' sequences are inserted into a plasmid in ''B. Subtillis'' expressing Tus, the replication fork arrest from the non-permissive
-end only occurs with 0.5% efficiency compared to 45.4% efficiency in a wild type ''E. coli'' system. If only Tus-''Ter'' interactions were important in the
+end only occurs with 0.5% efficiency compared to 45.4% efficiency in a wild type ''E. coli'' system<ref name = "Anderson"> Anderson, P., Griffith, A., Duggin, I. and Wake, R.  (2000) Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E.coli.  Molecular Microbiology, 36 (6): 1327-1335.</ref>. If only Tus-''Ter'' interactions were important in the
 mediation of polar fork arrest, then the efficiency in the two systems should be similar. This highlights the importance of other factors in the
 mediation of polar fork arrest.
@@ Line 53: / Line 52: @@
 <references/>
-Anderson, P., Griffith, A., Duggin, I. and Wake, R.  (2000) Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E.coli.  Molecular Microbiology, 36 (6): 1327-1335.
 Bastia, D., Zzaman, S., Krings, G., Saxena, M., Peng, X. and Greenberg, M.  (2008) Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase.  Proceedings of the National Academy of Science, USA, 105 (93): 12831-12836.
@@ Line 61: / Line 59: @@
-Mulcair, M. D., Schaeffer, P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, T. M. and Dixon, N .E.  (2006) A molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125: 1309-1319.
 Mulugu, S., Potnis, A., Shamsuzzaman, T. J., Alexander, K. and Bastia, D.  (2001) Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction.  Proceedings of the National Academy of Science, USA, 98 (17): 9569-9574.
 Neylon, C., Kralicek, A. V., Hill, T.M. and Dixon, N.E.  (2005) Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex.  Microbiology and Molecular Biology, 69 (3): 501-526.