RTP and Tus

A comparison of the Replication Terminator Protein (from Bacillus subtillis) and Tus (from Escherishia coli) provides an interesting insight into how proteins with vastly different structures and mechanisms of action can produce essentially identical effects in their native systems.

Looking at the structures of these two proteins, it is not immediately obvious that they would perfom the same function, specifically, to arrest the progression of the replication fork along the bacterial chromosome at specific sites (termed Ter sites). Furthermore, this arrest-mechanism functions in a polar manner in both organisms, which is perhaps surprising considering the symmetrical characteristics of both proteins.

Tus-Ter complex

The Replication Fork and Polar ArrestThe Replication Fork and Polar Arrest

DNA replication of circular bacterial chromosomes occurs using two replication forks that originate from one point (oriC), and move in opposite directions around the chromosome. In E. coli, B. Subtillis, and other bacteria and archaea, these replication forks are halted by interactions with terminator proteins bound to Ter sites. While it is possible for these organisms to not possess this type of replication-arrest mechanism, the conservation of this system across species indicates some sort of evolutionary benefit.

This Replication Fork Arrest mechanism was first studied using the Tus protein from Escherichia coli and the Replication Termination Protein (RTP) from Bacillus subtillis. Both of these proteins bind DNA sites known as "Terminator sites", or Ter sites. The termination of the replication fork is dependent on the direction of approach to these Ter sites: if the replication fork approaches from the permissive face replication will continue; however, if the replication fork approaches from the non-permissive face the fork will be arrested and DNA replication will cease at that point.

Tus: an asymmetric monomer, and unlikely candidate.Tus: an asymmetric monomer, and unlikely candidate.

In 1996, Kamada et al determined the crystal structure Tus bound to a 16bp fragment of Ter DNA. With no typical DNA-bidning motifs, Tus binds Ter DNA as an asymmetrical monomer, which establishes the basis for its polar arrest of the replication fork. Tus has three distinct regions: two α-helical regions and central β-strands which jointly form a large, positively-charged central cleft. The core β-structures embrace 13 base pairs of duplex DNA by partial insertion into the major groove, and at least 30 other residues make nonspecific contacts with the DNA backbone. The positioning of α-helices in the Tus protein is particularly interesting. Two protrude from both the amino and carboxy domains to clasp the DNA duplex, thereby shielding the interdomain β structures from direct contacts with other proteins (such as the DnaB helicase). The concentration of α-helices on the non-permissive face of Tus is absolutely cruical to the protein's ability to form a locked complex with the Ter site. More about this later!

Four models of replication-fork arrest

In a review paper in 2005, Neylon et al summarised the four proposed mechanisms of polar arrest by Tus-Ter complexes.

1. The Clamp model - Tus acts as a simple thermodynamic clamp which blocks DnaB progression.

2. The Interaction model - Tus directly interacts with DnaB to prevent its progression.

3. Tus engineers a DNA structure on the non-permissive face that is a physical block to DnaB

4. DnaB engineers a DNA structure on the permissive face that actively promotes Tus dissociation.

Neylon et al concluded that the Clamp model was too simplistic to explain the polar nature of fork arrest. They concluded based on mutational data that it is probably a combination of Tus-DnaB interactions as well as Tus-Ter binding strength that contribute to fork-arrest activity.

A stepwise model of dissociation of Tus from Ter DNA appealed to Neylon et al. This involved the formation of a nonspecific Tus-DNA complex before the formation of a specific Tus-Ter complex during binding. When DnaB approaches from the permissive end, it would promote the formation of the lower-affinity nonspecific complex, which would then rapidly dissociate. On approach to the nonpermissive face, formation of the nonspecific complex would be prevented, and Tus would become kinetically locked onto the Ter DNA.

So how does Tus actually stop the replication fork? And why is it a polar arrest mechanism?

Mulcair et al (2006) discovered that the key to Tus forming a locked complex with Ter was twofold: firstly, the locked complex was formed only on the approach of DnaB helicase (the leading edge of the replication fork), and secondly, this locked complex was due to the base-flipping of C6 of Ter DNA into a cytosine-specific binding pocket on Tus. The approach of DnaB is essential to lock formation as strand separation is required before the C6 base can twist out of the helix; this base is on the displaced strand, which explains why strand displacement by DnaB is halted. This C6 binds somewhere near the α4 helix, in or near the DNA-binding channel. Mulcair et al also showed that while this conformational change occurs at the non-permissive end of the complex when "locked", the interdomain and permissive face remain in a similar conformation to the non-locked complex.

His144 is a particularly important residue - it exists as its conjugate acid in the locked complex, forming hydrogen bonds with C6. Other residues - for example Phe140 and Gly149 - are also strictly conserved amongst different species' Tus protiens; many of the conserved residues among different Ter sites make base-specific contacts with Tus. The locked Tus-Ter complex is the most stable known monomeric DNA binding protein with a double-stranded sequence-specific recognition sequence - a half life of 550min has been reported (Mulcair, 2006). The formation of a large hydrogen-bond network is critical to sequence recognition and the stability of the twisted β-strands lying across the major groove.

When we thought we had a nice, elegant theory, someone had to come screw it all up.

In 2008, Bastia et al proposed that Tus is actually a polar antitranslocase, and that an AT-GC transversion at position 6 did not affect DnaB-translocation in vitro. They suggested that the base-flipping of C6 functions as a fail-safe mechanism, and that the replication fork is halted primarily by Tus-DnaB and Tus-Ter interactions.

spinqualitylabelsall models Tus complexed with Ter DNA (Kamada Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

spinqualitylabelsall models RTP complexed with Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

RTP: A homodimer responsible for Polar ArrestRTP: A homodimer responsible for Polar Arrest

The first crystal structure of the Replicator Terminator Protein (RTP) from Bacillus subtillis was determined in 1995 by Bussiere et al. This analysis revealed that RTP is comprised of two identical monomers, each of which binds to DNA to form a homodimer. The separate monomers bind at 30 bp sequences known as the A and B termination (Ter) sites. Both of these sites have inverted 16 bp repeats which overlap at highly conserved trinucleotide sequence. This first structure, which used a symmetric B Ter DNA homologue, suggested that the RTP exists as a symmetric homodimer. The idea that a symmetric protein structure could be responsible for an inherently polar mechanism has resulted in a series of proposed solutions and discoveries regarding the mechanism of replication fork arrest.

Differential Binding Affinity

The "Differential Binding Affinity Model", initially proposed by Kralicek et al. in 1997, states that the polar arrest mechanism can be explained purely based on the differential binding affinities of RTP to the A and B termination sites. The theory is based on the assumption that the affinity of RTP for the B site in the complete complex is much greater than the affinity of for the A site in the complete complex, or the affinity of a single RTP monomer to the B site alone. According to the model, only the affinity of the complex RTP for the B site (K3 in Figure X) is sufficient to prevent the removal of RTP from DNA when the replication fork moves along the DNA; therefore, if the replisome approaches from the B site, the RTP is not removed from the DNA and the replication fork is arrested. Similarly, when the replication fork approaches from the A site the binding affinity (K4 in Figure X) is not sufficient to prevent the removal of RTP and the replisome is able to pass.

The idea that RTP binds with differing affinity to the A and B Ter sites has since been explained on a molecular level with the determination of the crystal structure of RTP while bound to its native B Ter site (Vivian et al., 2007). This structure differed from that found by Bussiere et al. in that it used RTP bound to the native B Ter site, which is asymmetric, as opposed to a symmetric homologue. This revealed both the protein and the Ter DNA are asymmetric, potentially explaining the differential binding affinities between the A and B Ter sites.

This differential binding affinity model has since been refuted by mutational studies performed by Duggin et al. in 2004. After creating mutant DNA Ter sites and analysing the resulting efficiency of replication fork arrest, Duggin et al. found that mutations which caused decreased affinity of RTP for the proximal half of the terminator DNA (i.e. the half which faces the approaching replisome) did not necessarily decrease fork arrest efficiency, and that increased proximal site affinity did not increase fork arrest efficiency. These results were inconsistent with the differential binding affinity model, suggesting other factors must also be responsible for replication fork arrest by RTP.

Helicase Binding

The mutational data provided by Duggin et al. (2004) suggested that replication fork arrest was a more complex process than one based purely on binding affinities.

Comparison of Tus and RTPComparison of Tus and RTP

Include summarised table

Replication Fork Termination: The Future of DiscoveriesReplication Fork Termination: The Future of Discoveries

Relevance, studies into Eukaryotes.