Molecular analysis of cyanobacterial RNA polymerase genes

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Virginia Polytechnic Institute and State University

The RNA polymerase genes rpoBC1C2 (the rpoB and rpoC2 are incomplete) of the cyanobacterium Nostoc commune have been cloned, sequenced, and expressed both in vivo and in vitro using E. coli systems. The rpo genes encode large subunits of DNA-dependent RNA polymerase. Two genes in N. commune, rpoC1 and rpoC2, correspond to rpoC of E. coli, which indicates a divergent evolution of RNA polymerase. The rpoBC1C2genes of Nostoc are linked in the order of rpoB, rpoC1, and rpoC2, and are transcribed differently from the corresponding rpo genes of E. coli. In E. coli the rpoBC genes are co-transcribed, together with two ribosomal protein genes. The Nostoc rpoB gene is transcribed by one promoter while the rpoC1C2 genes are co-transcribed by another promoter. Northern analysis of Nostoc RNA revealed two transcripts (3.1 and 5.6 kb), which were specific for the rpoB and rpoC1C2 genes, respectively. SDS-PAGE, Coomassie staining and immunoanalysis detected two polypeptides with molecular weights of 72 and 94 kd when the cloned rpoBC1C2 fragment was expressed in E. coli systems. These two polypeptides were assigned as products of rpoC1 and rpC2, respectively. The transcription of RNA polymerase genes of N. commune is sensitive to water stress (drying). The rpo transcription ceases upon drying and resumes after the dried cells have been rewetted for more than 5 min. The RNA polymerase enzyme itself, however, is stable under the same drying conditions.