User:David Jung/BCHM3981 RTP Tus

From Proteopedia

Revision as of 13:54, 22 May 2011

The Replication Terminator Protein (RTP) is a protein involved in termination of replication in the gram positive bacterium, Bacillus subtilis. RTP was first identified in 1989, showing analogous function to Tus protein present in Escherichia coli (Lewis, Smith and Wake, 1989). Both RTP and Tus bind to termination sites (Ter sequences) present in the bacterial chromosome, terminating replication. Polar directionality of termination is assumed as circular bacterial chromosome is replicated bidirectionally with one replication fork going clockwise and the other one going anticlockwise. The RTP-Ter complex must therefore block a replication fork coming from one side but permit a fork from the opposite side. This is demonstrated in the Escherichia coli counterpart due to its apparent asymmetric structure.

Even though the protein is known to be involved in replication termination, its biological function is not well understood as mutants that lack certain Ter sites are shown to be viable in vitro (Iismaa and Wake. 1987). Thus, the biological functions of replication terminator proteins in bacteria have long been speculated. There are two hypotheses: inhibition of production of multimeric DNA, and post-initiation control of replication. Multimeric DNA is a dsDNA where multiple copies of the whole sequence are present. It has been shown, in the case of Tus in Escherichia coli, that without Tus-Ter interaction, it is more prone to overreplication (Hiasa and Marians, 1994). This is thought to be due to lack of inhibition of movement of DnaB, a helicase, along the replication fork by Tus-Ter complex. It was also shown that replication terminator protein is involved in post-initiation control of replication. The first level of control of replication was thought to occur before initiation. However, it has been experimentally determined that RTP-Ter complex maybe involved in second level of control after initiation to inhibit overreplication (Henckes et al., 1989).

Mechanism

For each terminator site (Ter site), two dimers of RTP bind. Each dimer binds to each half site present in a terminator site. The two half sites are termed A and B sites. Termination is observed to be blocked when the fork approaches the B site but not when it approaches the A site. However when B site was solely cloned into a vector, it could not effectively terminate replication, suggesting that induced polarity of RTP dimers on A and B site is required for termination (Smith and Wake, 1992). Many hypotheses and models have been proposed by a number of researchers as to how this biased replication termination can be carried out. Largely there have been two models: differential binding affinity model, and induced conformational change model.

Differential binding affinity model

Differential binding affinity model was proposed with the claim that the polarity is induced by binding of RTP dimers to each site with different strengths. It was hypothesised that the RTP dimer binding to the half site located in the "blocking" site binds tightly while the dimer binding to the "permissive" site binds less tightly to the site (Langly et al., 1993). However, after a series of experiments using mutant forms of terminator sites that contain RTP binding half sites with differential binding affinity, it was concluded that this differential binding affinity model cannot solely explain the polarity of termination (Duggin et al., 2005).

Structure

RTP is classified as a winged helix-loop-helix protein.

References

Duggin, I.G., Matthews, J.M., Dixon, N.E., Wake, R.G. and Mackay, J.P. (2005). A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Henckes, G., Harper, F., Levine, A., Vannier, F. and Seror, S.J. (1989). Overreplication of the origin region in the dnaB37 mutant of Bacillus subtilis: Postinitiation control of chromosomal replication. Proc. Natl. Acad. Sci. USA. 86: 8660-8664.

Hiasa, H. and Marians, K.J. (1994). Tus prevents overreplication of oriC plasmid DNA. The Journal of biological chemistry. 269(43):26959-26968.

Iiamaa, T.P. and Wake, R.G. (1987). The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation. J. Mol. Biol. 195:299-310.

Langly, D.B., Smith, M.T., Lewis, P.J. and Wake, R.G. (1993). Protein-nucleoside contacts in the interaction between the replication terminator protein of Bacillus subtilis and the DNA terminator. Molecular Microbiology. 10(4):771-779.

Lewis, P.J., Smith, M.T. and Wake, R.G. (1989). A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site. J. Bacteriology. 171(6):3564-3567.

Smith, M.T. and Wake, R.G. (1992). Definition and polarity of action of DNA replication terminators in Bacillus subtilis. J. Mol. Biol. 227(3):648-657.