RTP and Tus: Difference between revisions
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The Replication Terminator Protein (RTP) from ''Bacillus subtilis'' is comprised of two identical monomers 14.5 kDa in size which bind to DNA to form a homodimer. The separate monomers bind at 30 bp sequences known as the A and B termination (Ter) sites. Both of these sites have inverted 16 bp repeats which overlap at highly conserved TAT trinucleotide sequence. The structure of RTP is commonly referred to as a “winged helix” DNA binding motif and consists of a compact α helix / β-strand <scene name='RTP_and_Tus/Practice_structure/5'>secondary structure</scene> with a protruding loop (or “wing”) between the β2 and β3 strands. Both monomers of RTP interact with DNA specifically through hydrogen bonding at residues | The Replication Terminator Protein (RTP) from ''Bacillus subtilis'' is comprised of two identical monomers 14.5 kDa in size which bind to DNA to form a homodimer. The separate monomers bind at 30 bp sequences known as the A and B termination (Ter) sites. Both of these sites have inverted 16 bp repeats which overlap at highly conserved TAT trinucleotide sequence. The structure of RTP is commonly referred to as a “winged helix” DNA binding motif and consists of a compact α helix / β-strand <scene name='RTP_and_Tus/Practice_structure/5'>secondary structure</scene> with a protruding loop (or “wing”) between the β2 and β3 strands. Both monomers of RTP interact with DNA specifically through hydrogen bonding at residues | ||
<scene name='RTP_and_Tus/Practice_structure/7'>Arg 59, His 54 and Thr 55, and also through nonbonding contacts with Tyr 58</scene>, present in the α3 recognition helix. RTP also forms non-specific interactions at its N-terminus region.<ref>Wilce JA, Vivian JP, Hastings AF, Otting G, Folmer RHA, Duggin IG, Wake RG, Wilce MCJ (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature Structural Biology'' 8: 206-210.</ref> | <scene name='RTP_and_Tus/Practice_structure/7'>Arg 59, His 54 and Thr 55, and also through nonbonding contacts with Tyr 58</scene>, present in the α3 recognition helix. RTP also forms non-specific interactions at its N-terminus region.<ref>Wilce JA, Vivian JP, Hastings AF, Otting G, Folmer RHA, Duggin IG, Wake RG, Wilce MCJ (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature Structural Biology'' 8: 206-210.</ref> | ||
<scene name='RTP_and_Tus/Practice_structure/2'>(Reset Scene)</scene> | |||
The first crystal structure of RTP was determined in 1995 by Bussiere ''et al.'' (See figure above).<ref>Bussiere DE, Bastia D, White SW (1995) Crystal structure of the replication terminator protein from ''B. subtilis'' at 2.6 A. ''Cell'' 80(4): 651-60.</ref> This initial structure, which used a symmetric B ''Ter'' DNA homologue, suggested that the RTP exists as a symmetric homodimer. The idea that a symmetric protein structure could be responsible for an inherently polar mechanism has resulted in a series of proposed solutions and discoveries regarding the mechanism of replication fork arrest. | The first crystal structure of RTP was determined in 1995 by Bussiere ''et al.'' (See figure above).<ref>Bussiere DE, Bastia D, White SW (1995) Crystal structure of the replication terminator protein from ''B. subtilis'' at 2.6 A. ''Cell'' 80(4): 651-60.</ref> This initial structure, which used a symmetric B ''Ter'' DNA homologue, suggested that the RTP exists as a symmetric homodimer. The idea that a symmetric protein structure could be responsible for an inherently polar mechanism has resulted in a series of proposed solutions and discoveries regarding the mechanism of replication fork arrest. | ||
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<Structure load='2I06' size='400' frame='true' align='right' caption='Locked Tus-''Ter'' Complex (Mulcair ''et al.'' 2006)' scene='RTP_and_Tus/Co-ordination_of_his144/ | <Structure load='2I06' size='400' frame='true' align='right' caption='Locked Tus-''Ter'' Complex (Mulcair ''et al.'' 2006)' scene='RTP_and_Tus/Co-ordination_of_his144/8' /> | ||
== Tus: An asymmetric monomer and unlikely candidate. == | == Tus: An asymmetric monomer and unlikely candidate. == | ||
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Mulcair ''et al.'' (2006) discovered that the key to Tus forming a locked complex with ''Ter'' was twofold: firstly, the locked complex was formed only on the approach of DnaB helicase (the leading edge of the replication fork); and secondly, this locked complex was due to the base-flipping of C6 of ''Ter'' DNA into a cytosine-specific binding pocket on Tus. The approach of DnaB is essential to lock formation as strand separation is required before the C6 base can twist out of the helix; this base is located on the displaced strand, which explains why strand displacement by DnaB is halted. This C6 binds near the α4 helix, in or near the DNA-binding channel. Mulcair ''et al.'' also showed that while this conformational change occurs at the non-permissive end of the complex when "locked", the interdomain and permissive face remain in a similar conformation to the non-locked complex. | Mulcair ''et al.'' (2006) discovered that the key to Tus forming a locked complex with ''Ter'' was twofold: firstly, the locked complex was formed only on the approach of DnaB helicase (the leading edge of the replication fork); and secondly, this locked complex was due to the base-flipping of C6 of ''Ter'' DNA into a cytosine-specific binding pocket on Tus. The approach of DnaB is essential to lock formation as strand separation is required before the C6 base can twist out of the helix; this base is located on the displaced strand, which explains why strand displacement by DnaB is halted. This C6 binds near the α4 helix, in or near the DNA-binding channel. Mulcair ''et al.'' also showed that while this conformational change occurs at the non-permissive end of the complex when "locked", the interdomain and permissive face remain in a similar conformation to the non-locked complex. | ||
<scene name='RTP_and_Tus/Co-ordination_of_his144/7'>His144</scene> is a particularly important residue as 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 | <scene name='RTP_and_Tus/Co-ordination_of_his144/7'>His144</scene> is a particularly important residue as 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 proteins, and many of the conserved residues among different ''Ter'' sites make base-specific contacts with Tus. <scene name='RTP_and_Tus/Co-ordination_of_his144/8'>(Reset Scene)</scene> | ||
The locked Tus-''Ter'' complex is the most stable known monomeric DNA binding protein with a double-stranded sequence-specific recognition sequence, with a reported half life of 550min (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> | The locked Tus-''Ter'' complex is the most stable known monomeric DNA binding protein with a double-stranded sequence-specific recognition sequence, with a reported half life of 550min (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> | ||
