RTP and Tus: Difference between revisions
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[[Image:RTP_DBA_Model.jpg|300px|right|thumb| Differential Binding Affinity Model proposed by Kralicek ''et al.'' in 1997. | [[Image:RTP_DBA_Model.jpg|300px|right|thumb| Differential Binding Affinity Model proposed by Kralicek ''et al.'' in 1997. | ||
Image from Duggin ''et al.'', 2004. ]] | Image from Duggin ''et al.'', 2004. ]] | ||
'''Differential Binding Affinity''' | |||
In 1997, Kralicek ''et al.'' proposed an alternate theory of RTP arrest known as the '''Differential Binding Affinity Model'''. This model also involves the idea of a “molecular clamp”, and states that the polar arrest mechanism can be explained purely based on the differential binding affinities of RTP to the A and B termination sites. The theory is based on the assumption that the affinity of RTP for the B site in the complete complex is much greater than the affinity of for the A site in the complete complex, or the affinity of a single RTP monomer to the B site alone. According to the model, only the affinity of the complex RTP for the B site (K3 in Figure X) is sufficient to prevent the removal of RTP from DNA when the replication fork moves along the DNA; therefore, if the replisome approaches from the B site, the RTP is not removed from the DNA and the replication fork is arrested. Similarly, when the replication fork approaches from the A site the binding affinity (K4 in Figure X) is not sufficient to prevent the removal of RTP and the replisome is able to pass <ref>Kralicek, AV, Wilson PK, Ralston GB, Wake RG, King GF (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.</ref>. | In 1997, Kralicek ''et al.'' proposed an alternate theory of RTP arrest known as the '''Differential Binding Affinity Model'''. This model also involves the idea of a “molecular clamp”, and states that the polar arrest mechanism can be explained purely based on the differential binding affinities of RTP to the A and B termination sites. The theory is based on the assumption that the affinity of RTP for the B site in the complete complex is much greater than the affinity of for the A site in the complete complex, or the affinity of a single RTP monomer to the B site alone. According to the model, only the affinity of the complex RTP for the B site (K3 in Figure X) is sufficient to prevent the removal of RTP from DNA when the replication fork moves along the DNA; therefore, if the replisome approaches from the B site, the RTP is not removed from the DNA and the replication fork is arrested. Similarly, when the replication fork approaches from the A site the binding affinity (K4 in Figure X) is not sufficient to prevent the removal of RTP and the replisome is able to pass <ref>Kralicek, AV, Wilson PK, Ralston GB, Wake RG, King GF (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.</ref>. | ||
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The idea that RTP binds with differing affinity to the A and B Ter sites has since been explained on a molecular level with the determination of the crystal structure of RTP while bound to its native B Ter site by Vivian ''et al.'', 2007. This structure differed from that found by Bussiere ''et al.'' in that it used RTP bound to the native B Ter site, which is asymmetric, as opposed to a symmetric homologue. This revealed both the protein and the Ter DNA are asymmetric, potentially explaining the differential binding affinities between the A and B Ter sites <ref>Vivian JP, Porter CJ, Wilce JA, Wilce MCJ (2007) An asymmetric structure of the ''Bacillus subtilis'' Replication Terminator Protein in complex with DNA. ''Journal of Molecular Biology'' 370: 481-491.</ref>. | The idea that RTP binds with differing affinity to the A and B Ter sites has since been explained on a molecular level with the determination of the crystal structure of RTP while bound to its native B Ter site by Vivian ''et al.'', 2007. This structure differed from that found by Bussiere ''et al.'' in that it used RTP bound to the native B Ter site, which is asymmetric, as opposed to a symmetric homologue. This revealed both the protein and the Ter DNA are asymmetric, potentially explaining the differential binding affinities between the A and B Ter sites <ref>Vivian JP, Porter CJ, Wilce JA, Wilce MCJ (2007) An asymmetric structure of the ''Bacillus subtilis'' Replication Terminator Protein in complex with DNA. ''Journal of Molecular Biology'' 370: 481-491.</ref>. | ||
<Structure load='2EFW' size='400' frame='true' align='left' caption='RTP complexed with Ter DNA (Vivian ''et al'' 2007)' scene='Insert optional scene name here' /> | <Structure load='2EFW' size='400' frame='true' align='left' caption='RTP complexed with Ter DNA (Vivian ''et al'' 2007)' scene='Insert optional scene name here' />The entire concept of a “molecular clamp” in fork arrest has since been refuted by mutational studies performed by Duggin ''et al.'' in 2004. After creating mutant DNA Ter sites and analysing the resulting efficiency of replication fork arrest, Duggin ''et al.'' found that mutations which caused decreased affinity of RTP for the proximal half of the terminator DNA (i.e. the half which faces the approaching replisome) did not necessarily decrease fork arrest efficiency, and that increased proximal site affinity did not increase fork arrest efficiency. These results were inconsistent with the differential binding affinity model and induced conformational change, suggesting other factors apart from DNA-protein binding must also be responsible for replication fork arrest by RTP <ref>Duggin IG, Matthews JM, Dixon, NE, Wake RG, Mackay JP (2004) A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of ''Bacillus subtilis''. ''The Journal of Biological Chemistry'' 280(13): 13105-13113.</ref>. | ||
'''Helicase Binding''' | '''Helicase Binding''' | ||
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The 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. To identify an alternate model, in 2009 Duggin created a series of RTP fusion proteins which were constructed by adding GFP to the C-terminal with a variety of spacer amino acids. <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> | The 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. To identify an alternate model, in 2009 Duggin created a series of RTP fusion proteins which were constructed by adding GFP to the C-terminal with a variety of spacer amino acids. <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> | ||
TO BE CONTINUED!! | TO BE CONTINUED!! | ||
