2002 Fiscal Year Final Research Report Summary
Molecular mechanismn of transcription termination factor Rho as a hexameric RAN/DNA helicase
| Project/Area Number |
13680762
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SHIGESADA Katsuya Kyoto Univ., Inst. Virus Res., Assoc Prof., ウイルス研究所, 助教授 (40009626)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Rho factor / Transcription termination / F1-ATPase homology / Helicase / Troidal hexamer / Chemical cross-linking / 3-D structure / X-ray crystallography |
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| Research Abstract |
The E. coli transcription termination protein Rho is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid in an ATPdependent manner. To further elucidate the molecular mechanism of Rho, we have focused on its structural similarity to F1-ATP-ase and conducted the following two lines of studies
1) Our previous cross-linking study suggested that the Rho hexamer has a pseudo-C3 symmetry, in which its subunits take dual conformanonal states in an alternating manner. We further tried herein to map the positions of cross-links on Rho by digestion with hydroxylamine, which singly cuts the rho polypeptide between Asn 151 and Gly 152. The result revealed that a lysine on the N-terminal fragment from one subunit is cross-linked to a lysine on the C-terminal fragment from an adjacent subunit at all subunit-subunit interfaces. On the other band, a three-dimensional reconstruction of Rho hexamer predicted that Lysl23 is positioned in close proximity to Lys224 and Lys249 on an adjacent subunit. Thus, Lysl23 would make a good candidate for crosslinks to Lys224 or Lys249
2) Toward determining the exact three-dimensional structure of Rho, we also set out for its X-ray crystallographic analysis. Initially, we could only obtain Rho crystals of poor qualities that showed no clear diffractions. Through subsequent repeated trials, we have identified several conditions that could improve the quality of Rho crystal: a) addition of a high concentration of KC1 (0 5 M); b) a prolonged aging of crystal; c) methylation of lysine residues; and so on. Even with these conditions combined, however, best crystals so far made yielded diffractions at unsatisfactory resolutions of at most 10 Angstrom. Most recently, we have found that the addition of ligands such as ATP analogs and oligo (dC) help to maintain Rho protein in a mono-disperse state, a prerequisite for making good crystals. Thus trials are in progress to crystallize complexes containing such ligands
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