|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
(1) To solve the interaction between PhoB and RNA polymerase in transcription activation of the Pho regulon, we isolated RNA polymerase mutants which cause specific defect in transcription activation of the pho regulon. The mutations were mapped in the sigma-70 subunit of RNA polymerasae. In vitro and in vivo experiments using the mutant RNA polymerases indicated that the interaction site between PhoB and RNA polymerase is located in the sigma-70 subunit. The result was also supported by the experiment based on the site-directed mutagenesis of the sigma-70 subunit of RNA polymerase.
(2) We succeeded the isolation of the new Pho gene, phoH,of which expression is dependent on PhoB.Indentificaiton of the region of the promoter and the gene product, and functional analysis of the gene product were performed.
(3) We releaved that acetyl-phosphate, a high-enargy small intermediate, can phosphorylate PhoB.The form of PhoB phosphorylated by acetyl-phosphate is fully active in transcription activation of th Pho regulon as the form that is phosphorylated by the congnate kinases, PhoR and PhoM.
(4) To reveal the function of the extream heat shock sigma factor, sigma-24, we constructed a strain that has a deletion of the sigma-24 gene. The strain is leathal at high temperature. The result indicates that the sigma-24 is essential to growth at high temperature.
(5) The sigma-38 factor that is specific in stational phase of bacterial growth. To identify the specific sequences of the statianl phase specific promoters, we isolated a lot of the mutant promoters, which are not recognized by the RNA polymerase containing the sigma-38, by PCR mutagenesis. With futher analyzes, we conclude that the RNA polymerase containing the sigma-38 recongnizes the-10 sequence, but does not recognize the-35 sequence.