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 pol ymerase (RNAP) that contains the sigma(70)) was purified and character ized using two classical sigma(70) type promotes, the bacteriophage T7 A1 and the RNA I promoters. Transcription from these promoters was sen sitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed ag ainst the Escherichia coli sigma(70) subunit). Specific transcripts we re detected in vitro for R. capsulatus cytochrome c(2) (cycA) and fruc tose-inducible (fruB) promoters and genes induced in photosynthesis (p uf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma(70) indi cated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. c apsulatus nifA1 promoter, which exhibits only three of the five consen sus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed n o transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma(70) en zyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.