User:Nathan Harris/Tus: Difference between revisions

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.   

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 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.  

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. 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 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 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 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.