The E.coli protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ter sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA []. Unlike RTP termination sites, the ten E.coli ter sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [8]. The tus-ter complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression [1].
Tus structure
Tus is a member of the replication termination protein family and has no similarity to any DNA-binding motif that is known, and is organized into two discontinuous domains (N terminal and
C terminal) that consist of α helical and β sheets. Two pairs of antiparallel β strands link the N and C terminal protein domains and form the DNA-binding interdomain. This provides a large, positivley charged, central cleft into which the double helix (deformed locally) can fit, so that the two α helical domains flank the DNA. The interdomain β strands, which makes up the core region of the 13bp DNA-binding, that sit across from the DNA, accesses a deepened major groove and contacts several of the bases within this groove, and is responsible for ter sequence recognition and binding to DNA. There are 17 sequence-specific interactions between the DNA and the α helixes and β strands presented by Tus, although the majority of these interactions are from the proximinal β sheets. The helicase-blocking or non-permissive face, of Tus consists of α helices and loops from N and C terminal domains. Tus is unrelated structurally to the replication termination protein despite their similar functions.
Tus Mechanism of Action
Two models were proposed to explain the mechanism of Tus activity generated by early experiments. The "clamp model" proposed that the Tus-ter complex created a barrier that arrested the progression of the replication machinery from one direction but not the other, by DNA binding [12]. The "interaction model" suggested that a particular region of Tus specifically interacted with the progressing helicase, causing it to halt the fork, and this interaction would only be possible at one face of the protein [13]. The structure of the tus-DNA complex has recently been solved [8]. It suggests that the protein can block helicase approaching from one direction and not the other, without the necessity of specific Tus–helicase interactions. The Tus-Ter complex could act as a physical barrier against the replication fork at the non-permissive face; the α helical regions protrude from the protein around the DNA and block the helicase from accessing the region tightly bound to the DNA. On the other hand, when the helicase advances from the opposite direction it does not encounter the α helical barriers and can disrupt Tus-DNA binding by interrupting the interdomain β strands, causing Tus to be released. This simple model is supported by studies where mutants were screened after exhibiting a reduction in their ability to arrest replication [8]. Most of the mutations occurred in the interdomain β-strands and none of these mutations occurred in the blocking surface that may contact the progressing helicase [8]. However it is important to note that a specific interaction between the blocking face and the helicase cannot be ruled out based on structural studies, and that it if present it may have a role to enhance the physical barrier’s effectiveness.
