RTP and Tus: Difference between revisions
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A comparison of the Replication Terminator Protein (from ''Bacillus subtilis'') and Tus (from ''Escherichia coli'') provides an interesting insight into how proteins with vastly different structures and mechanisms of action can produce essentially identical effects in their native systems. | A comparison of the Replication Terminator Protein (from ''Bacillus subtilis'') and Tus (from ''Escherichia coli'') provides an interesting insight into how proteins with vastly different structures and mechanisms of action can produce essentially identical effects in their native systems. | ||
Looking at the structures of these two proteins, it is not immediately obvious that they would perfom the same function | Looking at the structures of these two proteins, it is not immediately obvious that they would perfom the same function; to arrest the progression of the replication fork along the bacterial chromosome at specific sites (''Ter'' sites). Furthermore, this arrest-mechanism functions in a polar manner in both organisms, which is perhaps surprising considering the symmetrical characteristics of both proteins.<ref>Wake, RG and King, GF (1997) A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. ''Structure'' 5: 1-5.</ref> | ||
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DNA replication of circular bacterial chromosomes occurs using two replication forks that originate from | DNA replication of circular bacterial chromosomes occurs using two replication forks that originate from a single location (''oriC'') and move in opposite directions around the chromosome. In ''E. coli'', ''B. subtilis'', and other bacteria and archaea, these replication forks are halted by interactions with terminator proteins bound to DNA sites known as "Terminator sites", or''Ter'' sites. The termination of the replication fork is dependent on the direction of approach to these ''Ter'' sites: if the replication fork approaches from the permissive face replication will continue; however, if the replication fork approaches from the non-permissive face the fork will be arrested and DNA replication will cease at that point. While it is possible for these organisms to function without this type of replication-arrest mechanism, the conservation of this system across species indicates some form of evolutionary benefit. | ||
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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 | 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>. 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>. 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> | ||
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 | 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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'''Is replication fork arrest by RTP affinity independent?''' | '''Is replication fork arrest by RTP affinity independent?''' | ||
This mutational data provided by Duggin ''et al.'' (2004) suggested that replication fork arrest was a more complex process than one based purely on binding between RTP and DNA. In 2009, Duggin proposed the alternate theory that the C-terminus of an RTP monomer is able to contact the oncoming helicase and that this interaction is responsible for replication fork arrest. To test this hypothesis, Duggin created a series of RTP fusion proteins which were constructed by adding a GFP peptide to the C-terminus using a variety of spacer amino acids. Because the C-terminus is not in contact with DNA, the fusion proteins had no affect on the DNA binding properties of RTP; however, the fusion proteins had significantly reduced fork arrest efficiencies. These results effectively rule out the molecular clamp model, showing that RTP activity is independent of DNA binding affinity and suggesting that this C-terminal interaction with helicase is responsible for the fork arrest properties of RTP. <ref>Duggin, IG (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: 1-6.</ref> | This mutational data provided by Duggin ''et al.'' (2004) suggested that replication fork arrest was a more complex process than one based purely on binding between RTP and DNA. In 2009, Duggin proposed the alternate theory that the C-terminus of an RTP monomer is able to contact the oncoming helicase and that this interaction is responsible for replication fork arrest. To test this hypothesis, Duggin created a series of RTP fusion proteins which were constructed by adding a GFP peptide to the C-terminus using a variety of spacer amino acids. Because the C-terminus is not in contact with DNA, the fusion proteins had no affect on the DNA binding properties of RTP; however, the fusion proteins had significantly reduced fork arrest efficiencies. These results effectively rule out the molecular clamp model, showing that RTP activity is independent of DNA binding affinity and suggesting that this C-terminal interaction with helicase is responsible for the fork arrest properties of RTP.<ref>Duggin, IG (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: 1-6.</ref> | ||
