| Co-Investigator(Kenkyū-buntansha) |
SOGAWA Kumiko National Institute of Genetics, Structural Biology Center, Assistant, 構造遺伝学研究センター, 助手 (20291073)
NAGAI Hiroki National Institute of Genetics, Structural Biology Center, Assistant, 構造遺伝学研究センター, 助手 (80222173)
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| Research Abstract |
All bacteria have major sigma factors, which enable RNA polymerase to recognize most of their promoters, and σィイD170ィエD1 is the major sigma factor of E. coli. The largest finding in this research is the aggregation of σィイD170ィエD1 in response to environment such as high physiological temperature and the end of log phase. The purified σィイD170ィエD1 also makes aggregates at similar temperature, while a mutant protein with deletion of aa 130-374 exists as a mixture of monomer and aggregates at all temperatures. The monomeric mutant protein is as active as the wild-type σィイD170ィエD1 in purified reconstituted transcription system, while oligomers have at most 15% of the activity. Thus the domain of aa 130-374 is not essential for transcription reaction but has essential role to prevent aggregation of σィイD170ィエD1. We have first succeeded to constructed a disruptant of rpoD (the gene of σィイD170ィエD1) and the mutant σィイD170ィエD1 and SigA, the major sigma of B. subtilis lacking aa 130-374, complement the disruption in limited conditions, proving that aa 130-374 is not required for minimal transcription in vivo.
By cleaving σィイD170ィエD1 with hydroxide radical, the exposed surfaces of σィイD170ィエD1 were identified in monomeric and aggregated forms. The regions protected upon aggregation sandwich the regions exposed upon aggregation, suggesting the existence of more than two interacting sites for aggregation.
We have determined the role of omega subunit of E. coli RNA polymerase. The core enzyme prepared from omega-depleted cells binds GroEL shaperonin, and the removal of GroEL destroys the ability of core enzyme to bind σィイD170ィエD1. This proves the role of omega is to maturate core enzyme, and explain why the depletion of omega does not bring any phenotype. This proves the role of omega is to maturate core enzyme, and explain why the depletion of omega does not bring any phenotype.
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