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.
The Replication Fork and Polar ArrestThe Replication Fork and Polar Arrest
In circular bacterial chromosomes, DNA replication occurs using two replication forks which move along the chromosome in opposite directions. To increase the efficiency of this process, the replication forks are stopped at ...
This Replication Fork Arrest mechanism was first studied using the Tus protein from Escherichia coli and the Replication Termination Protein (RTP) from Basillus 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 replication will cease.
...
Tus:...Tus:...
The structure of Tus is unusual for a DNA-binding protein. It 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 (Kamada, 1996). 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!
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 C6 binds somewhere near the α4 helix, in or near the DNA-binding channel.
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.
|
|
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.