TRANSCRIPTION OF THE RHODOBACTER-SPHAEROIDES CYCA P1 PROMOTER BY ALTERNATE RNA-POLYMERASE HOLOENZYMES

These experiments sought to identify what form of RNA polymerase trans cribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c(2) gene (cycA), In vitro, cycA P1 was recognized by an RNA polymerase ho loenzyme fraction that transcribes several well-characterized Escheric hia coli heat shock (sigma(32)) promoters, The in vivo effects of muta tions banking the transcription initiation site (+1) also suggested th at cycA P1 was recognized by an RNA polymerase similar to E. coli E si gma(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 poin t mutation at position -34 that is towards the E. coli E sigma(32) -35 consensus sequence (G34T) increased cycA P1 activity similar to 20-fo ld, while several mutations that reduced or abolished promoter functio n changed highly conserved bases in presumed -10 or -35 elements, In a ddition, cycA P1 function was retained in mutant promoters with a spac er region as short as 14 nucleotides. When either wild-type or G34T pr omoters 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 fami ly (E sigma(37)) or a 38-kDa subunit that also allows core RNA polymer ase to recognize E. coli heat shock promoters (E sigma(38)) (R. K. Kar ls, J, Brooks, P. Rossmeissl, J. Luedke, and T, J, Donohue, J. Bacteri ol, 180:10-19, 1998).