RTP and Tus: Difference between revisions
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<scene name='RTP_and_Tus/Practice_structure/3'> | The Replication Terminator Protein (RTP) from ''Bacillus subtilis'' is comprised of two identical dimers, each of which binds to DNA to form a homodimer. The separate dimers 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/3'>secondary structure</scene> with a protruding loop (or “wing”) between the β2 and β3 strands. Both dimers of RTP interact with DNA specifically through hydrogen bonding of residues Arg 59, His 54 and Thr 55, and also through nonbonding contacts with Tyr 58. 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 analysis revealed that RTP is This first 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. | |||
'''Induced Conformational Change''' | '''Induced Conformational Change''' | ||
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[[Image:RTP_DBA_Model2.jpg|300px|right|thumb| Differential Binding Affinity Model proposed by Kralicek ''et al.'' in 1997. | [[Image:RTP_DBA_Model2.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. ]] | ||
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 | 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 dimer 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>. | ||
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>. | ||
The entire concept of a “molecular clamp” in fork arrest has since been | The entire concept of a “molecular clamp” in fork arrest has since been challenged 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>. | ||
