User:Karen Lee/Sandbox 1
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===Key Features of RTP=== | ===Key Features of RTP=== | ||
| - | The | + | The functional RTP-DNA complex requires two dimers of RTP. Dimerisation occurs through the extended <scene name='User:Karen_Lee/Sandbox_1/Alpha4_helix_interaction/1'>α4-helix</scene> of each monomer forming an anti-parallel coiled coil. It is thought that upon binding to the ''Ter'' sequence, the complex is able to impede the progress of replicative machinery headed by the replicative helicase. This is supported by ''in vitro'' studies involving ''E. coli'' where RTP was shown to interact with the DnaB helicase, thus a similar interaction may also occur in ''B. subtilis'' <ref>Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. (2001) A Single Domain of the Replication Termination Protein of ''Bacillus subtilis'' Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. ''J. Biol. Chem.'' '''276(26)''', 23471-23479.</ref>. |
| - | Initial | + | Initial studies of the RTP-DNA structure were performed on a ''TerI'' (the first ''Ter'' site that the clockwise replication fork encounters) B site (sRB) which was symmetric in sequence<ref name="Wilce">Wilce, J. A., Vivian, J. P., Hastings, A. F., Otting, G., Folmer, R. H., Duggin, I. G. ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature Struct. Biol.'' '''8''', 206-210.</ref>. NMR and crystallography revealed a symmetric RTP bound to the symmetric DNA site. However, further studies on the native ''TerI'' B site (nRB) showed an asymmetric structure. 6 base pair differences between the sRB and nRB define the native site: three upstream and three downstream, leading to an asymmetric RTP binding to the site. What is of importance is that the RTP-sRB interaction showed similar binding affinity to the RTP-nRB interaction, but had reduced fork arrest capability<ref name="Wilce" />. |
This differential capability is due to the location of the 6 base pair changes. The three downstream changes have no bearing on the conformation of RTP as no base-specific interactions are made in this region. However, the three upstream changes are located within the major groove of the dsDNA of the ''TerI'' site. This is where the <scene name='User:Karen_Lee/Sandbox_1/Rtp_alpha_helix_3_dna/2'>α3 helix</scene> binds which underlies RTP binding specificity<ref name="Vivian" />. This affects the binding of the RTP monomer to the upstream region and initiates the structural difference observed. | This differential capability is due to the location of the 6 base pair changes. The three downstream changes have no bearing on the conformation of RTP as no base-specific interactions are made in this region. However, the three upstream changes are located within the major groove of the dsDNA of the ''TerI'' site. This is where the <scene name='User:Karen_Lee/Sandbox_1/Rtp_alpha_helix_3_dna/2'>α3 helix</scene> binds which underlies RTP binding specificity<ref name="Vivian" />. This affects the binding of the RTP monomer to the upstream region and initiates the structural difference observed. | ||
Revision as of 04:35, 15 May 2011
Contents |
Replication Termination
Replication is an essential process in all cells. The process copies the chromosomal DNA of the organism to provide the extra copy needed in cell division and is therefore critical in the biological inheritance of genes. In cells with circular chromosomes, replication starts from a single origin proceed with two replication forks moving in opposite directions. It should follow that this process must be terminated or multiple copies of the chromosome would be made.
The process of terminating replication is performed by replication termination proteins. These proteins bind to specific sequences in the DNA, called Ter sites. This binding provides a physical blockage in the DNA that stops the replication machinery. In B. subtilis, the termination protein is called Replication Terminator Protein (RTP), and in E. coli it is Termination Utilisation Substance (Tus).
In each circular chromosome, there are two sets of Ter sites that appear roughly opposite to the origin of replication. One set blocks the clockwise replication fork while the other traps the anti-clockwise replication fork. Interestingly, both these proteins bind to DNA in such a way that they terminate the replication fork in one direction, but simultaneously allow the replication fork to continue in the other direction.
RTP
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Structure of RTP
A single RTP monomer consists of four , three and an unstructured domain. The binds to DNA by into the major groove of Ter sites, while the interacts with the minor groove. The N-terminal arm also binds to the Ter site[1].
RTP conforms to the classic winged-helix motif, a compact α/β structure with αβααββ topology where "" project from the loop between and [2]. However, each monomer lacks a well-formed β1-strand (named β1-loop) and has an additional following the β3-strand[3].
(.)
Key Features of RTP
The functional RTP-DNA complex requires two dimers of RTP. Dimerisation occurs through the extended of each monomer forming an anti-parallel coiled coil. It is thought that upon binding to the Ter sequence, the complex is able to impede the progress of replicative machinery headed by the replicative helicase. This is supported by in vitro studies involving E. coli where RTP was shown to interact with the DnaB helicase, thus a similar interaction may also occur in B. subtilis [4].
Initial studies of the RTP-DNA structure were performed on a TerI (the first Ter site that the clockwise replication fork encounters) B site (sRB) which was symmetric in sequence[5]. NMR and crystallography revealed a symmetric RTP bound to the symmetric DNA site. However, further studies on the native TerI B site (nRB) showed an asymmetric structure. 6 base pair differences between the sRB and nRB define the native site: three upstream and three downstream, leading to an asymmetric RTP binding to the site. What is of importance is that the RTP-sRB interaction showed similar binding affinity to the RTP-nRB interaction, but had reduced fork arrest capability[5].
This differential capability is due to the location of the 6 base pair changes. The three downstream changes have no bearing on the conformation of RTP as no base-specific interactions are made in this region. However, the three upstream changes are located within the major groove of the dsDNA of the TerI site. This is where the binds which underlies RTP binding specificity[3]. This affects the binding of the RTP monomer to the upstream region and initiates the structural difference observed.
This leads into the fact that RTP binds asymmetrically across the nRB site and therefore allows the complex to act as a polar barrier to the replication fork. When the fork approaches the B site (with tight RTP-DNA binding) the fork is unable to progress and is paused. Approaching from the A site does not impede its progress. This polarity can be explained by both the differential binding affinity as explained above and the cooperative binding affect of the RTP monomers. The complex formed between an RTP dimer and the B site facilitates cooperative binding of another RTP dimer to the A site to form a complete RTP-Ter complex[3].
RTP as a checkpoint regulator
An interesting feature is that RTP can cause replication fork pausing in B. subtilis at regions other than Ter sites. There appear to be stringent terminator (STer) regions where RTP can bind and arrest replication. These regions are located around 200 kb either side of the origin of replication[6]. Studies have shown that there is 76% homology between this site and the normal Ter sites[7]. Additionally, there is no need for two dimers to interact in vitro, which avoids unwanted arrest during normal replication.
Tus
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Structure of Tus
The Tus protein works as a monomer, each monomer consists of two domains, the amino and carboxy domains. The overall structure of Tus is made up of two separated by central twisted anti-parallel . These structures together form a that accommodates a B-form Ter . This binding involves two β-strands involes contacting the bases and sugar-phosphate back bones from the major groove of the Ter DNA. Upon Tus binding, the major groove becomes deeper and the minor groove is significantly deeper, causing the DNA to be slightly bent.
References
- ↑ Pai, S. K., Bussiere, D. E., Wang, F., Hutchinson, C. A., White, S. W. & Bastia, D. (1996) The structure and function of the replication terminator protein of Bacillus subtilis: identification of the ‘winged helix’ DNA-binding domain. EMBO J. 15(12), 3164-3173.
- ↑ Gajiwala, K. S. & Burley, S. K. (2000) Winged Helix proteins. Curr. Opin. Struct. Biol. 10, 110-116.
- ↑ 3.0 3.1 3.2 Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. (2007) An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. J. Mol. Biol. 370, 481-491.
- ↑ Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. (2001) A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276(26), 23471-23479.
- ↑ 5.0 5.1 Wilce, J. A., Vivian, J. P., Hastings, A. F., Otting, G., Folmer, R. H., Duggin, I. G. et al. (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nature Struct. Biol. 8, 206-210.
- ↑ Levine, A., Vannier, F., Dehbi, M., Henckes, G. & Seror, S. J. (1991) The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli. J. Mol. Biol. 219, 605-613.
- ↑ Autret, S., Levine, A., Vannier, F., Fujita, Y. & Seror, S. J. (1999) The replication checkpoint control in Bacillus subtilis: identification of a novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response. Mol. Microbiol., 31, 1665-1679.
