Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides

Abstract

We have been characterizing RNA polymerase holoenzymes from Rhodobacter sphaeroides. RNA polymerase purified from R. sphaeroides transcribed from promoters recognized by Escherichia coli Eσ32 or Eσ70 holoenzyme. Antisera to E. coli σ32 or σ70 indicated that related polypeptides of ~37 kDa (σ37) and 93 kDa (σ93), respectively, are present in this preparation. Transcription of σ32-dependent promoters was observed in a further fractionated R. sphaeroides holoenzyme containing the σ37 polypeptide, while a preparation enriched in σ93 transcribed σ70- dependent promoters. To demonstrate further that the σ93 polypeptide functions like E. coli σ70, we obtained an R. sphaeroides Eσ93 holoenzyme capable of transcription from σ70-dependent promoters by combining σ93 with (i) an Eσ37 fraction with diminished σ93 polypeptide content or (ii) E. coli core RNA polymerase. The generation of analogous DNase I footprints on the lacUV5 promoter by R. sphaeroides Eσ93 and by E. coli Eσ70 suggests that the overall structures of these two holoenzymes are similar. However, some differences in promoter specificity between R. sphaeroides Eσ93 and E. coli Eσ70 exist because transcription of an R. sphaeroides rRNA promoter was detected only with Eσ93.