| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
SUMMARV OF RBSEARCK RESULTS Three species of Escherichia coli a factors, σ70, σ38, and 054, were engineered so as to have a unique cysteine residue at 1 2, 9, and 5 different positions, respectively. The mutant σ factors were individually modified, at the unique cystein residue, with a protein/DNA cleavage agent (FeBABE). Tethered 0 derivatives were then used to reconstitute holoenzyme and transcription initiation complex, and proximity sites on the p and p1 subunits and the template DNA were analyzed after contact-dependent cleavage of protein and DNA. Our results suggest that all three sigma factors interact with the core enzyme and the template DNA in almost similar manner, although they are significantly different in the size and structure. I Fragments of P' subunit covering various evolutionary conserved regions were prepared and analyzed for their binding to 070 factor in vitro. Together with the result of FeBABE experiments, we conclude that the N-terminal proximal region of the p1 subunit between residues 201 and 345 is the primary binding site for 070. Eleven σ70 mutants with alanine-substitutions in the region 4.1 were used to analyze the role of"this highly conserved region. In vitro transcription and DNase I footprinting results suggest that several amino acid residues on the conserved region 4.1 are critical, probably through direct interaction with core enzyme subunit, for presenting conserved region 4.2 (the DNA-binding helices) toward the -35 region of promoter DNA. All seven 0 factors of Escherichia coli were purified and compared for their binding affinities to the core enzyme. The results, together with the measurement of intracellular concentration of each σ factor, let us estimate the relative abundance of each holoenzyme species in the cell.
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