User:Nathan Harris/Tus: Difference between revisions

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'''Tus''' is a DNA binding protein involved in the termination of bi-directional replication in ''Escherichia coli''. Tus binds specifically to ''Ter'' sequences within the ''E. Coli'' genome forming a Tus- ''Ter'' complex which functions to trap replication forks. Tus binds to ''Ter'' sites as an asymmetric monomer creating a permissive and non-permissive face to allow for polar fork arrest.

'''Tus''' is a DNA binding protein involved in the termination of bi-directional replication in ''Escherichia coli''. Tus binds specifically to ''Ter'' sequences within the ''E. Coli'' genome forming a Tus- ''Ter'' complex which functions to trap replication forks. Tus binds to ''Ter'' sites as an asymmetric monomer creating a permissive and non-permissive face to allow for polar fork arrest <ref name = "Neylon"> 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.

</ref>.

----

=='''Biological role'''==

Multiple ''Ter'' sites (''TerA''- ''TerJ'') are located in regions destined for replication termination in ''E. coli''. Tus binds specifically to these 23bp ''Ter'' sites forming a Tus-''Ter'' complex. This complex allows for the blocking of an approaching replication fork in one direction, the non-permissive face, but not from the other direction, the permissive face. The ability to halt the replication machinery at the non-permissive face is thought to involve the inhibition of DnaB Helicase, preventing it from unwinding DNA. DnaB inhibition has been proposed to occur either through protein-protein interactions between Tus and DnaB, or by a physical block provided by Protein-DNA interactions i.e. the Tus-''Ter'' complex. Recent models suggest a potentially combination of these two mechanisms. Evolution of this termination system has allowed for efficient replication by ''E. coli'' as it prevents any over expenditure of energy or time. Different replication proteins have been found in other model organisms, such as RTP in ''Bacillus subtilis''. Despite similar biological roles of RTP and Tus they have significantly different structures.

Multiple ''Ter'' sites (''TerA''- ''TerJ'') are located in regions destined for replication termination in ''E. coli''. Tus binds specifically to these 23bp ''Ter'' sites forming a Tus-''Ter'' complex . This complex allows for the blocking of an approaching replication fork in one direction, the non-permissive face, but not from the other direction, the permissive face. The ability to halt the replication machinery at the non-permissive face is thought to involve the inhibition of DnaB Helicase, preventing it from unwinding DNA. DnaB inhibition has been proposed to occur either through protein-protein interactions between Tus and DnaB, or by a physical block provided by Protein-DNA interactions i.e. the Tus-''Ter'' complex <ref name = "Kamada"> Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto, N. and Morikawa, K. (1996) Structure of a replication-terminator protein complexed with DNA. Nature 383 (6681): 598-603.</ref>. Recent models suggest a potentially combination of these two mechanisms <ref name = "Kaplan"> Kaplan, D. L. and Bastia, D. (2009) Mechanisms of polar arrest of replication fork. Molecular biology, 72 (2): 279-285.</ref>. Evolution of this termination system has allowed for efficient replication by ''E. coli'' as it prevents any over expenditure of energy or time. Different replication proteins have been found in other model organisms, such as RTP in ''Bacillus subtilis''. Despite similar biological roles of RTP and Tus they have significantly different structures <ref name = "Kaplan" />.

~~----~~

=='''Ter sites'''==

The Tus protein binds as a monomer through several direct and indirect contacts to conserved ''Ter'' sites. Ter sites are signified by 23 bp of consensus sequences which maintain a highly conserved C6 and 13 bp core region that interacts with Tus. Additionally, ''Ter'' sites are arranged in groups of five located opposite to the origin of replication. Within each group the ''Ter'' sites have a coordinated polarity of termination.

The Tus protein binds as a monomer through several direct and indirect contacts to conserved ''Ter'' sites. Ter sites are signified by 23 bp of consensus sequences which maintain a highly conserved C6 and 13 bp core region that interacts with Tus. Additionally, ''Ter'' sites are arranged in groups of five located opposite to the origin of replication. Within each group the ''Ter'' sites have a coordinated polarity of termination <ref name = "Neylon" />.

~~----~~

=='''Structure of Tus protein and binding interactions with TerA'''==

{{STRUCTURE_1ecr| PDB=1ecr | SCENE=User:Nathan_Harris/Tus/Opening_scene/1}}Tus constitutes 308 amino acids and a mass of approximately 36 kDa. The structural components of Tus have been elucidated through crystal structures of Tus bound to <scene name='User:Nathan_Harris/Tus/Ter/1'>TerA</scene> <ref name = "Kamada" />. Tus exhibits a unique binding motif to Ter sites previously undescribed from any known protein-DNA interactions.

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/2'>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 = "Neylon" /><ref name = "Kamada" />.

Tus constitutes 308 amino acid residues and a mass of approximately 36 kDa. The structural components of Tus have been elucidated through crystal structures of Tus bound to a fragment of ''TerA''. Tus exhibits a unique binding motif to Ter sites previously undescribed from any known protein-DNA interactions.

Tus is divided into an amino and carboxy domain distinguished by two alpha helical regions and central β sheets combining to encompass a large central basic cleft. The interdomain region consists of anti-parallel β strands and an extended L4 loop which connect the amino and carboxy domains. Within this interdomain region, the βF, βG, βH and βI 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 (αIV and αV) which also contribute to the hydrophobic core of the protein. Within the β sandwich, βCADKE contacts the alpha helical region, whereas βLFIJ 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 L3 loop is responsible for connecting helices αVI and αVII and also contacts the minor groove of DNA.

~~----~~

=='''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 ~~20o~~. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site.

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'''===

===='''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

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 C6 of Ter.

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 hydrophobic pocket ~~(G149, H144, I79, F140)~~ of Tus forming a so called locked complex.

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.

This locking results in a dramatic increase in the affinity of Tus for Ter. In contrast, the progressive unwinding of Ter from the permissive face

This locking results in a dramatic increase in the affinity of Tus for ''Ter''. In contrast, the progressive unwinding of ''Ter'' from the permissive face

results in dissociation of Tus from Ter. It is interesting to note that this C6 is conserved amongst all Ter sequences, further demonstrating the

results in dissociation of Tus from ''Ter''. It is interesting to note that this C6 is conserved amongst all ''Ter'' sequences, further demonstrating the

likelihood of its importance in replication arrest.

This leads to a model suggesting that DnaB approaching from the non-permissive face unwinds Ter until it reaches the C6. When C6 is unwound it flips

This leads to a model suggesting that DnaB approaching from the non-permissive face unwinds ''Ter'' until it reaches the C6. When C6 is unwound it flips

to form a locked complex with Tus hence preventing any further progression of the replication machinery, i.e. a physical block to the DnaB. However

when the DnaB approaches from the permissive face, the C6 is located at the opposite end of the Ter sequence and so is unable to form a locked complex

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

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.

Tus-DnaB interactions

Numerous studies support a model for replication termination resulting specifically from Tus-DnaB protein interactions.

==='''Tus-DnaB interactions'''===

Experimentation in the field has demonstrated that the E49 within the L1 loop of the non-permissive face of Tus is important in the formation of protein-protein interactions with DnaB. When this glutamic acid is exchanged for lysine (E49K), an increase in affinity for Ter and a decrease in affinity for DnaB result. Despite the increased affinity for Ter, this E49K mutatation results in a reduced capability of polar replication fork termination demonstrating the importance of Tus-DnaB interactions.

Numerous studies support a model for replication termination resulting specifically from Tus-DnaB protein interactions. Experimentation in the field has demonstrated that the <scene name='User:Nathan_Harris/Tus/E49/1'>E49</scene> within the L1 loop of the non-permissive face of Tus is important in the formation of protein-protein interactions with DnaB. When this glutamic acid is exchanged for lysine (E49K), an increase in affinity for ''Ter'' and a decrease in affinity for DnaB result <ref name = "Henderson"> Henderson, T., Niles, A., Valjavec-Gratian, M. and Hill, T. (2001) Site-directed mutagenesis and phylogenetic comparisons of Escherichia coli Tus protein: DNA-protein interactions alone cannot account for Tus activity. Molecular Genetics and Genomics, 265 (6): 941-953.</ref><ref name = "Mulugu"> 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.</ref>. Despite the increased affinity for ''Ter'', this E49K mutatation results in a reduced capability of polar replication fork termination demonstrating the importance of Tus-DnaB interactions.

In further confirmation of this helicase specific mechanism, the engineering of intra-strand covalent crosslinks introduced immediately upstream of the C6 of Ter prevent DnaB helicase from unwinding the C6. Despite this inability to unwind and from a locked complex with Tus, polar fork termination is still permitted indicating that the formation of a locked complex is unnecessary for replication termination.

In further confirmation of this helicase specific mechanism, the engineering of intra-strand covalent crosslinks introduced immediately upstream of the C6 of ''Ter'' prevent DnaB helicase from unwinding the C6 <ref name = "Bastia"> 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.</ref>. Despite this inability to unwind and from a locked complex with ''Tus'', polar fork termination is still permitted indicating that the formation of a locked complex is unnecessary for replication termination.

Current Models

Recent models for the termination of replication in E. coli propose that when DnaB approaches the Tus-Ter complex from the permissive face there are no considerable protein-protein interactions between the DnaB and Tus resulting in the dislodgement of Tus from Ter and hence allowing for the progression of the replication fork. However, when DnaB approaches the non-permissive face, significant protein-protein interactions between the DnaB and Tus prevent the dislodgement of Tus, resulting in replication termination. If for any reason this mechanism may fail, DnaB will unwind Ter until it reaches C6 which would induce the formation of a locked complex and subsequent prevention of replication fork progression.

==='''Current Models'''===

References

Recent models for the termination of replication in ''E. coli'' propose that when DnaB approaches the Tus-''Ter'' complex from the permissive face there are no considerable protein-protein interactions between the DnaB and Tus resulting in the dislodgement of Tus from ''Ter'' and hence allowing for the progression of the replication fork. However, when DnaB approaches the non-permissive face, significant protein-protein interactions between the DnaB and Tus prevent the dislodgement of Tus, resulting in replication termination. If for any reason this mechanism may fail, DnaB will unwind ''Ter'' until it reaches C6 which would induce the formation of a locked complex and subsequent prevention of replication fork progression <ref name = "Kaplan" />.

~~Mulcair, M. D., Schaeffer P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, M. H & Dixon, N. E.~~

=='''References'''==

@@ Line 1: / Line 1: @@
-'''Tus''' is a DNA binding protein involved in the termination of bi-directional replication in ''Escherichia coli''. Tus binds specifically to ''Ter'' sequences within the ''E. Coli'' genome forming a Tus- ''Ter'' complex which functions to trap replication forks. Tus binds to ''Ter'' sites as an asymmetric monomer creating a permissive and non-permissive face to allow for polar fork arrest.
+'''Tus''' is a DNA binding protein involved in the termination of bi-directional replication in ''Escherichia coli''. Tus binds specifically to ''Ter'' sequences within the ''E. Coli'' genome forming a Tus- ''Ter'' complex which functions to trap replication forks. Tus binds to ''Ter'' sites as an asymmetric monomer creating a permissive and non-permissive face to allow for polar fork arrest <ref name = "Neylon"> 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.
+</ref>.
-----
 =='''Biological role'''==
-Multiple ''Ter'' sites (''TerA''- ''TerJ'') are located in regions destined for replication termination in ''E. coli''. Tus binds specifically to these 23bp ''Ter'' sites forming a Tus-''Ter'' complex. This complex allows for the blocking of an approaching replication fork in one direction, the non-permissive face, but not from the other direction, the permissive face.  The ability to halt the replication machinery at the non-permissive face is thought to involve the inhibition of DnaB Helicase, preventing it from unwinding DNA. DnaB inhibition has been proposed to occur either through protein-protein interactions between Tus and DnaB, or by a physical block provided by Protein-DNA interactions i.e. the Tus-''Ter'' complex.  Recent models suggest a potentially combination of these two mechanisms. Evolution of this termination system has allowed for efficient replication by ''E. coli'' as it prevents any over expenditure of energy or time.  Different replication proteins have been found in other model organisms, such as RTP in ''Bacillus subtilis''.  Despite similar biological roles of RTP and Tus they have significantly different structures.
+Multiple ''Ter'' sites (''TerA''- ''TerJ'') are located in regions destined for replication termination in ''E. coli''. Tus binds specifically to these 23bp ''Ter'' sites forming a Tus-''Ter'' complex . This complex allows for the blocking of an approaching replication fork in one direction, the non-permissive face, but not from the other direction, the permissive face.  The ability to halt the replication machinery at the non-permissive face is thought to involve the inhibition of DnaB Helicase, preventing it from unwinding DNA. DnaB inhibition has been proposed to occur either through protein-protein interactions between Tus and DnaB, or by a physical block provided by Protein-DNA interactions i.e. the Tus-''Ter'' complex <ref name = "Kamada"> Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto, N. and Morikawa, K.  (1996) Structure of a replication-terminator protein complexed with DNA.  Nature 383 (6681): 598-603.</ref>.  Recent models suggest a potentially combination of these two mechanisms <ref name = "Kaplan"> Kaplan, D. L. and Bastia, D.  (2009) Mechanisms of polar arrest of replication fork.  Molecular biology, 72 (2): 279-285.</ref>. Evolution of this termination system has allowed for efficient replication by ''E. coli'' as it prevents any over expenditure of energy or time.  Different replication proteins have been found in other model organisms, such as RTP in ''Bacillus subtilis''.  Despite similar biological roles of RTP and Tus they have significantly different structures <ref name = "Kaplan" />.
-----
 =='''Ter sites'''==
-The Tus protein binds as a monomer through several direct and indirect contacts to conserved ''Ter'' sites.  Ter sites are signified by 23 bp of consensus sequences which maintain a highly conserved C6 and 13 bp core region that interacts with Tus.  Additionally, ''Ter'' sites are arranged in groups of five located opposite to the origin of replication.  Within each group the ''Ter'' sites have a coordinated polarity of termination.
+The Tus protein binds as a monomer through several direct and indirect contacts to conserved ''Ter'' sites.  Ter sites are signified by 23 bp of consensus sequences which maintain a highly conserved C6 and 13 bp core region that interacts with Tus.  Additionally, ''Ter'' sites are arranged in groups of five located opposite to the origin of replication.  Within each group the ''Ter'' sites have a coordinated polarity of termination <ref name = "Neylon" />.
-----
 =='''Structure of Tus protein and binding interactions with TerA'''==
+{{STRUCTURE_1ecr| PDB=1ecr | SCENE=User:Nathan_Harris/Tus/Opening_scene/1}}Tus constitutes 308 amino acids and a mass of approximately 36 kDa. The structural components of Tus have been elucidated through crystal structures of Tus bound to <scene name='User:Nathan_Harris/Tus/Ter/1'>TerA</scene> <ref name = "Kamada" />. Tus exhibits a unique binding motif to Ter sites previously undescribed from any known protein-DNA interactions.
+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/2'>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 = "Neylon" /><ref name = "Kamada" />.
-Tus constitutes 308 amino acid residues and a mass of approximately 36 kDa. The structural components of Tus have been elucidated through crystal structures of Tus bound to a fragment of ''TerA''. Tus exhibits a unique binding motif to Ter sites previously undescribed from any known protein-DNA interactions.
-Tus is divided into an amino and carboxy domain distinguished by two alpha helical regions and central β sheets combining to encompass a large central basic cleft. The interdomain region consists of anti-parallel β strands and an extended L4 loop which connect the amino and carboxy domains. Within this interdomain region, the βF, βG, βH and βI 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 (αIV and αV) which also contribute to the hydrophobic core of the protein. Within the β sandwich, βCADKE contacts the alpha helical region, whereas βLFIJ 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 L3 loop is responsible for connecting helices αVI and αVII and also contacts the minor groove of DNA.
-----
 =='''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 20o. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate ''Ter'' site.
+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'''===
+===='''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
+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 C6 of Ter.
+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 hydrophobic pocket (G149, H144, I79, F140) of Tus forming a so called locked complex.
+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.
-This locking results in a dramatic increase in the affinity of Tus for Ter. In contrast, the progressive unwinding of Ter from the permissive face
+This locking results in a dramatic increase in the affinity of Tus for ''Ter''. In contrast, the progressive unwinding of ''Ter'' from the permissive face
-results in dissociation of Tus from Ter. It is interesting to note that this C6 is conserved amongst all Ter sequences, further demonstrating the
+results in dissociation of Tus from ''Ter''. It is interesting to note that this C6 is conserved amongst all ''Ter'' sequences, further demonstrating the
 likelihood of its importance in replication arrest.
-This leads to a model suggesting that DnaB approaching from the non-permissive face unwinds Ter until it reaches the C6. When C6 is unwound it flips
+This leads to a model suggesting that DnaB approaching from the non-permissive face unwinds ''Ter'' until it reaches the C6. When C6 is unwound it flips
 to form a locked complex with Tus hence preventing any further progression of the replication machinery, i.e. a physical block to the DnaB. However
 when the DnaB approaches from the permissive face, the C6 is located at the opposite end of the Ter sequence and so is unable to form a locked complex
 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
+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.
-Tus-DnaB interactions
-Numerous studies support a model for replication termination resulting specifically from Tus-DnaB protein interactions.
+==='''Tus-DnaB interactions'''===
-Experimentation in the field has demonstrated that the E49 within the L1 loop of the non-permissive face of Tus is important in the formation of protein-protein interactions with DnaB. When this glutamic acid is exchanged for lysine (E49K), an increase in affinity for Ter and a decrease in affinity for DnaB result. Despite the increased affinity for Ter, this E49K mutatation results in a reduced capability of polar replication fork termination demonstrating the importance of Tus-DnaB interactions.
+Numerous studies support a model for replication termination resulting specifically from Tus-DnaB protein interactions. Experimentation in the field has demonstrated that the <scene name='User:Nathan_Harris/Tus/E49/1'>E49</scene> within the L1 loop of the non-permissive face of Tus is important in the formation of protein-protein interactions with DnaB. When this glutamic acid is exchanged for lysine (E49K), an increase in affinity for ''Ter'' and a decrease in affinity for DnaB result <ref name = "Henderson"> Henderson, T., Niles, A., Valjavec-Gratian, M. and Hill, T.  (2001) Site-directed mutagenesis and phylogenetic comparisons of Escherichia coli Tus protein: DNA-protein interactions alone cannot account for Tus activity.  Molecular Genetics and Genomics, 265 (6): 941-953.</ref><ref name = "Mulugu"> 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.</ref>. Despite the increased affinity for ''Ter'', this E49K mutatation results in a reduced capability of polar replication fork termination demonstrating the importance of Tus-DnaB interactions.
-In further confirmation of this helicase specific mechanism, the engineering of intra-strand covalent crosslinks introduced immediately upstream of the C6 of Ter prevent DnaB helicase from unwinding the C6. Despite this inability to unwind and from a locked complex with Tus, polar fork termination is still permitted indicating that the formation of a locked complex is unnecessary for replication termination.
+In further confirmation of this helicase specific mechanism, the engineering of intra-strand covalent crosslinks introduced immediately upstream of the C6 of ''Ter'' prevent DnaB helicase from unwinding the C6 <ref name = "Bastia"> 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.</ref>. Despite this inability to unwind and from a locked complex with ''Tus'', polar fork termination is still permitted indicating that the formation of a locked complex is unnecessary for replication termination.
-Current Models
-Recent models for the termination of replication in E. coli propose that when DnaB approaches the Tus-Ter complex from the permissive face there are no considerable protein-protein interactions between the DnaB and Tus resulting in the dislodgement of Tus from Ter and hence allowing for the progression of the replication fork. However, when DnaB approaches the non-permissive face, significant protein-protein interactions between the DnaB and Tus prevent the dislodgement of Tus, resulting in replication termination. If for any reason this mechanism may fail, DnaB will unwind Ter until it reaches C6 which would induce the formation of a locked complex and subsequent prevention of replication fork progression.
+==='''Current Models'''===
-References
+Recent models for the termination of replication in ''E. coli'' propose that when DnaB approaches the Tus-''Ter'' complex from the permissive face there are no considerable protein-protein interactions between the DnaB and Tus resulting in the dislodgement of Tus from ''Ter'' and hence allowing for the progression of the replication fork. However, when DnaB approaches the non-permissive face, significant protein-protein interactions between the DnaB and Tus prevent the dislodgement of Tus, resulting in replication termination. If for any reason this mechanism may fail, DnaB will unwind ''Ter'' until it reaches C6 which would induce the formation of a locked complex and subsequent prevention of replication fork progression <ref name = "Kaplan" />.
-Mulcair, M. D., Schaeffer P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, M. H & Dixon, N. E.
+=='''References'''==
+<references/>