RTP and Tus: Difference between revisions
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In 1996, Kamada ''et al.'' determined the crystal structure Tus bound to a 16bp fragment of ''Ter'' DNA. With no typical DNA-bidning motifs, Tus 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. 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. | In 1996, Kamada ''et al.'' determined the crystal structure Tus bound to a 16bp fragment of ''Ter'' DNA. With no typical DNA-bidning motifs, Tus 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. 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)<ref>Kamada | 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).<ref>Kamada K, Horiuchi T, Ohsumi K, Shimamoto N, Morikawa K (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' 383: 598 - 603.</ref> 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! | ||
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Neylon ''et al.'' concluded that the Clamp model was too simplistic to explain the polar nature of fork arrest. They concluded based on mutational data that it is probably a combination of Tus-DnaB interactions as well as Tus-''Ter'' binding strength that contribute to fork-arrest activity. | Neylon ''et al.'' concluded that the Clamp model was too simplistic to explain the polar nature of fork arrest. They concluded based on mutational data that it is probably a combination of Tus-DnaB interactions as well as Tus-''Ter'' binding strength that contribute to fork-arrest activity. | ||
A stepwise model of dissociation of Tus from ''Ter'' DNA appealed to Neylon ''et al''. This involved the formation of a nonspecific Tus-DNA complex before the formation of a specific Tus-''Ter'' complex during binding. When DnaB approaches from the permissive end, it would promote the formation of the lower-affinity nonspecific complex, which would then rapidly dissociate. On approach to the nonpermissive face, formation of the nonspecific complex would be prevented, and Tus would become kinetically locked onto the ''Ter'' DNA.<ref>Neylon | A stepwise model of dissociation of Tus from ''Ter'' DNA appealed to Neylon ''et al''. This involved the formation of a nonspecific Tus-DNA complex before the formation of a specific Tus-''Ter'' complex during binding. When DnaB approaches from the permissive end, it would promote the formation of the lower-affinity nonspecific complex, which would then rapidly dissociate. On approach to the nonpermissive face, formation of the nonspecific complex would be prevented, and Tus would become kinetically locked onto the ''Ter'' DNA.<ref>Neylon C, Kralicek AV, Hill TM, Dixon NE (2005) Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex. ''Microbiology and Molecular Biology Reviews'' 69: 501 - 526. </ref> | ||
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<scene name='RTP_and_Tus/Co-ordination_of_his144/7'>His144</scene> 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. | <scene name='RTP_and_Tus/Co-ordination_of_his144/7'>His144</scene> 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.<ref>Mulcair | |||
</ref> | 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 ''et al.'', 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.<ref>Mulcair MD, Schaeffer PM, Oakley AJ, Cross HF, Neylon C, Hill TM, Dixon NE (2006) A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. ''Cell'' 125: 1309 - 1319.</ref> | ||
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'''Just when we thought we had a nice, elegant theory...''' | '''Just when we thought we had a nice, elegant theory...''' | ||
In 2008, Bastia ''et al.'' proposed that Tus is actually a polar antitranslocase, and that an AT-GC transversion at position 6 did not affect DnaB-translocation ''in vitro''. They suggested that the base-flipping of C6 functions as a fail-safe mechanism, and that the replication fork is halted primarily by Tus-DnaB and Tus-''Ter'' interactions.<ref>Bastia | In 2008, Bastia ''et al.'' proposed that Tus is actually a polar antitranslocase, and that an AT-GC transversion at position 6 did not affect DnaB-translocation ''in vitro''. They suggested that the base-flipping of C6 functions as a fail-safe mechanism, and that the replication fork is halted primarily by Tus-DnaB and Tus-''Ter'' interactions.<ref>Bastia D, Zzaman S, Krings G, Saxena M, Peng XH, Greenberg MM (2008) Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase. ''Proceedings of the National Academy of Sciences'' 105: 12831 - 12836.</ref> | ||
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== Comparison of Tus and RTP == | == Comparison of Tus and RTP == | ||
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== Replication Fork Termination: The Future of Discoveries == | == Replication Fork Termination: The Future of Discoveries == | ||
Perhaps surprisingly, replication fork termination has also been identified in specific regions of eukaryotes chromosoms. In the yeast ''Saccharomyces cerevisiae'' the terminator protein Fob1p has been shown to arrest the replication fork at ''Ter'' sites in non-transcribed spacer regions of rDNA; while in ''Schizosaccharomyes pombe'' polar replication termination by RTS1 has been shown to control the direction of replication of the ''mat1'' mating locus, enabling the yeast to alternate between mating types. This provides evidence that the replication termination process has been adapted by a wide range of organisms and is able to perform a variety of functions, opening up an exciting new field of research. <ref>Kaplan DL, Bastia D (2009) Mechanisms of polar arrest of a replication fork. ''Molecular Microbiology'' 72(2): 279-285.</ref> | Perhaps surprisingly, replication fork termination has also been identified in specific regions of eukaryotes chromosoms. In the yeast ''Saccharomyces cerevisiae'' the terminator protein Fob1p has been shown to arrest the replication fork at ''Ter'' sites in non-transcribed spacer regions of rDNA; while in ''Schizosaccharomyes pombe'' polar replication termination by RTS1 has been shown to control the direction of replication of the ''mat1'' mating locus, enabling the yeast to alternate between mating types. This provides evidence that the replication termination process has been adapted by a wide range of organisms and is able to perform a variety of functions, opening up an exciting new field of research. <ref>Kaplan DL, Bastia D (2009) Mechanisms of polar arrest of a replication fork. ''Molecular Microbiology'' 72(2): 279-285.</ref> | ||
== References == | == References == | ||
<references/> | <references/> | ||
