User:David Jung/BCHM3981 RTP Tus
From Proteopedia
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| - | == | + | == Structure == |
| - | + | <Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Insert optional scene name here' /> | |
| + | The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3). | ||
| - | '''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). | ||
| - | '''Induced conformational change model''' | ||
| - | Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b). | ||
| - | == Structure == | ||
| - | <Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Insert optional scene name here' /> | ||
| - | The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3). | ||
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| + | == 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). | ||
| + | |||
| + | '''Induced conformational change model''' | ||
| + | Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b). | ||
Revision as of 03:12, 23 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).
Structure
|
The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian et al., 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian et al., 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian et al., 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna et al., 1996b; Pai et al., 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (sRB) used in the study (Wilce et al., 2001) (Fig 2). Later, Vivian et al. was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP TerI B site (nRB) (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian et al., 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3).
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).
Induced conformational change model Kralicek et al. proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek et al., 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek et al., 1997). In Vivian et al.’s X-ray crystallography work on RTP.C110S: nRB complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian et al., 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna et al., 1996b).
References
Duggin, I. G. (2006). DNA replication fork arrest by the Bacillus subtilis RTP-DNA complex involves a mechanism that is independent of the affinity of RTP-DNA binding. Journal of Molecular Biology 361(1), 1-6.
Duggin, I. G., Andersen, P. A., Smith, M. T., Wilce, J. A., King, G. F., and Wake, R. G. (1999). Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest. Journal of Molecular Biology 286(5), 1325-1335.
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. Journal of Biological Chemistry 280(13), 13105-13113.
Griffiths, A. A., Andersen, P. A., and Wake, R. G. (1998). Replication terminator protein-based replication fork-arrest systems in various Bacillus species. Journal of Bacteriology 180(13), 3360-3367.
Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto, N., and Morikawa, K. (1996). Structure of a replication-terminator protein complexed with DNA. Nature 383(6601), 598-603.
Kralicek, A. V., Wilson, P. K., Ralston, G. B., Wake, R. G., and King, G. F. (1997). Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex. Nucleic Acids Research 25(3), 590-596.
Langley, 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.
Lee, E. H., and Kornberg, A. (1992). Features of replication fork blockage by the Escherichia coli terminus binding protein. Journal of Biological Chemistry 267(13), 8778-8784.
Lee, E. H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989). Escherichia coli replication termination protein impedes the action of helicases. Proceedings of the National Academy of Sciences of the United States of America 86(23), 9104-9108.
Lewis, P. J., Ralston, G. B., Christopherson, R. I., and Wake, R. G. (1990). Identification of the replication terminator protein-binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding. Journal of Molecular Biology 214(1), 73-84.
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.
Manna, A. C., Pai, K. S., Bussiere, D. E., Davies, C., White, S. W., and Bastia, D. (1996a). Helicase-contrahelicase interaction and the mechanism of termination of DNA replication. Cell 87(5), 881-891.
Manna, A. C., Pai, K. S., Bussiere, D. E., White, S. W., and Bastia, D. (1996b). The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication. Proceedings of the National Academy of Sciences of the United States of America 93(8), 3253-3258.
Mohanty, B. K., Sahoo, T., and Bastia, D. (1998). Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa. Journal of Biological Chemistry 273(5), 3051-3059.
Pai, K. S., Bussiere, D. E., Wang, F. G., Hutchison, C. A., White, S. W., and Bastia, D. (1996). The structure and function of the replication terminator protein of Bacillus subtilis: Identification of the 'winged helix' DNA-binding domain. Embo Journal 15(12), 3164-3173.
Sahoo, T., Mohanty, B. K., Lobert, M., Manna, A. C., and Bastia, D. (1995). The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding. Journal of Biological Chemistry 270(49), 29138-29144.
Smith, M. T., deVries, C. J., Langley, D. B., King, G. F., and Wake, R. G. (1996). The Bacillus subtilis DNA replication terminator. Journal of Molecular Biology 260(1), 54-69.
Smith, M. T., Langley, D. B., Young, P. A., and Wake, R. G. (1994). The minimal sequence needed to define a functional dna terminator in Bacillus subtilis. Journal of Molecular Biology 241(3), 335-340.
Smith, M. T., and Wake, R. G. (1992). Definition and polarity of action of DNA replication terminators in Bacillus subtilis. Journal of Molecular Biology 227(3), 648-657.
Vivian, J. P., Porter, C. J., Wilce, J. A., and Wilce, M. C. J. (2007). An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA. Journal of Molecular Biology 370(3), 481-491.
Wake, R. G. (1997). Replication fork arrest and termination of chromosome replication in Bacillus subtilis. Fems Microbiology Letters 153(2), 247-254.
Wilce, J. A., Vivian, J. P., Hastings, A. F., Otting, G., Folmer, R. H. A., Duggin, I. G., Wake, R. G., and Wilce, M. C. J. (2001). Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nature Structural Biology 8(3), 206-210.
