Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

Abstract

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c 2 gene ( cycA ). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (ς 32 ) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς 32 . Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coli Eς 32 −35 consensus sequence (G34T) increased cycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς 37 ) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς 38 ) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).