User:Nathan Harris/Tus: Difference between revisions

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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 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.

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 C6 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 hydrophobic pocket (G149, H144, I79, F140) 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. 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

==='''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 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.

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.

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. 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.

----

=='''References'''==

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

@@ Line 29: / Line 29: @@
 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 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.
-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 C6 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 hydrophobic pocket (G149, H144, I79, F140) 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. 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
+==='''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 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.
+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.
-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. 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.
+----
+=='''References'''==
 Mulcair, M. D., Schaeffer P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, M. H & Dixon, N. E.