Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase - In vitro transcription of photosynthesis and other genes

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma(70)) was purified and characterized using two classical sigma(70) type promotes, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli sigma(70) subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c(2) (cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma(70) indicated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma(70) enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.