View source for User:David Jung/BCHM3981 RTP Tus

'''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 ''et al.'' 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 allow the replication fork to pass unimpeded from the “permissive” direction, while halting the fork from the “blocking” direction (Griffiths, Andersen, and Wake, 1998). This is demonstrated in the ''Escherichia coli'' counterpart due to its apparent asymmetric structure (Kamada ''et al.'', 1996). However, this asymmetry is not observed in RTP's 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). 

[[Image:B. subtilis 9 Ter sites.jpg |thumb|alt= It shows ''Ter'' sites within ''Bacillus subtilis'' |Fig 1. Replication termination (''Ter'') sites in ''Bacillus subtilis'']]

== ''Ter'' sites == 
[[Image:Fig 2.jpg |thumb|left|upright=1.5|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]
B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992). 





















== Structure ==
[[Image:Fig 3.jpg |thumb|left|upright=1.3|alt= Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.|Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.]]

<Structure load='2EFW' size='300' frame='true' align='right' caption='2 RTP dimers bound to Ter half sites' 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).

<applet size='200' frame='true' align='left' caption='RTP monomer' scene='User:David_Jung/BCHM3981_RTP_Tus/Monomer/2'/>
<applet size='200' frame='true' align='left' caption='RTP dimer' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp_dimer/1'/>
<applet size='200' frame='true' align='left' caption='RTP:sRB complex' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp-srb_complex/2'/>

[[Image:Fig 4.1.jpg |thumb|left|upright=1.6|alt= Fig 4.1 ''TerI'' site from ''B. subtilis'' showing the nRB sequence (blue)|Fig 4.1 ''TerI'' site from ''B. subtilis'' showing the nRB sequence (blue)]]

[[Image:Fig 5.jpg |thumb|upright=1.6|alt= Fig 5. Structural differences between the RTP.C110S:nRB and RTP.C110S:sRB complexes. (a) RTP.C110S crystal structures from nRB (blue) and sRB (yellow) complexes superimposed upon one another (from “top view”). The angle between α2 and α3 is indicated for each subunit. (b) Plot of root mean square deviation (RMSD) values versus residue number for each α-carbon position of the two RTP.C110S:nRB monomers (wing-up and wing-down) compared with each other (green) and with the RTP.C110S:sRB monomer structure (wing-up, red and wing-down, black).|Fig 5. Structural differences between the RTP.C110S:nRB and RTP.C110S:sRB complexes. (a) RTP.C110S crystal structures from nRB (blue) and sRB (yellow) complexes superimposed upon one another (from “top view”). The angle between α2 and α3 is indicated for each subunit. (b) Plot of root mean square deviation (RMSD) values versus residue number for each α-carbon position of the two RTP.C110S:nRB monomers (wing-up and wing-down) compared with each other (green) and with the RTP.C110S:sRB monomer structure (wing-up, red and wing-down, black).]]

[[Image:Fig 4.2.jpg |thumb|left|upright=2.0|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]










































































== Mechanism ==
[[Image:Fig 8.jpg |thumb|right|upright=2.0|alt= Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.|Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.]]

It appears that cooperative binding at both A and B sites are required for fork arrest. It has been demonstrated that binding at B site is a pre-requisite to A site binding (Langley ''et al.'', 1993) and binding of B site alone on a vector is not sufficient to stop the advancing  helicase (Smith ''et al.'', 1996; Smith ''et al.'', 1994; Smith and Wake, 1992). Only when both half-sites are filled (K<sub>D</sub> = 3 x 10<sup>-11</sup> M) that fork arrest occurs (Duggin ''et al.'', 1999).
Many hypotheses and models have been proposed by a number of researchers to explain this polar mechanism for replication termination (Duggin ''et al.'', 2005; Kralicek ''et al.'', 1997; Manna ''et al.'', 1996a; Manna ''et al.'', 1996b; Wake, 1997). Overall, there have been two models: the differential binding affinity model and the induced conformational change model, both proposed by Kralicek ''et al.'' in 1997 (Kralicek ''et al.'', 1997).



[[Image:Fig 6.jpg |thumb|left|upright=1.0|alt= RTP fusion proteins have normal binding affinity at ''TerI''. Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.|RTP fusion proteins have normal binding affinity at ''TerI''. Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.]]

[[Image:Fig 7.jpg |thumb|left|upright=1.0|alt= RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis''. (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).|RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis''. (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).]]

'''Differential binding affinity model'''

Differential binding affinity (aka the “directional molecular clamp” in E. coli (Lee and Kornberg, 1992; Lee ''et al.'', 1989; Mohanty, Sahoo, and Bastia, 1998; Sahoo ''et al.'', 1995)) model was proposed with the claim that the polarity is induced by binding of RTP dimers to each site with different strengths (Fig 8). 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 (Langley ''et al.'', 1993). Duggin ''et al.'' carried out a series of experiments which supported cooperative binding. When a complete ''TerI'' sequence had its ''nRB'' site substituted with ''sRB'', there was a fivefold reduction in the cooperative binding of the A site and a 50% reduction in fork arrest. Further substitutions of A and B sites with ''sRB'' found that there mutant sequences resulted in reduction in fork arrest despite similar binding affinities (Duggin ''et al.'', 2005). Furthermore, Duggin experimented with RTP fusion proteins consisting of extra peptides (GFP, 10, 20 or 40 residues long) attached to the C-terminus of α4-helix which was predicted to contact the approaching replisome. In vivo, these fusion proteins showed normal RTP-DNA binding affinity on the gel shift assay, but Southern Blot showed that replication fork arrest efficiency was reduced, not directly proportional to peptide size or sequence (Duggin, 2006) (Fig 6, 7). These experiments suggest that differential binding affinity cannot be the sole explanation for the polar fork arrest. However Duggin’s experiment also implies that specific interactions of RTP α4-helix with helicase is not the sole contributor in fork arrest either, as ~40% fork arrest activity was still present despite fusion peptide interference (Fig 7b).


'''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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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. <br />
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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. <br />