Abstract
To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma70 factor (R. capsulatus RNAP/sigma70) was purified and characterized using two classical sigma70 type promoters, 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 sigma70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c2 (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/sigma70 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/sigma70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.
Highlights
As elucidated by many in vitro studies during the last 30 years, bacterial transcription requires a core RNA polymerase (RNAP)1 enzyme and factors that recognize specific promoter elements
The R. capsulatus RNAP protein was purified by PEG precipitation followed by heparin-agarose and DEAE-Sepharose chromatography to at least 95% homogeneity by SDS-PAGE analysis (Fig. 1)
The ratio of intensity of the R. capsulatus RNAP ␣ polypeptides to the  and Ј bands correspond to 2:1:1 stoichiometry, similar to the E. coli RNAP (Fig. 1, compare lane 3 with lane 6)
Summary
As elucidated by many in vitro studies during the last 30 years, bacterial transcription requires a core RNA polymerase (RNAP) enzyme and factors that recognize specific promoter elements. Bch and puc are thought to be transcribed by the R. capsulatus RNA polymerase/70 holoenzyme, based on promoter sequences, it is unclear what factor(s) recognize the puf and puh genes. Light-dependent stimulation of transcription from the puf and puh operons requires the hvrA gene [10]. The oxygen-regulated puf, puh, and puc operons require the regA/regB-encoded two-component system, it is unknown whether these promoters are directly activated by such proteins [11, 12]. Results of in vitro transcription studies on these mutant promoters confirmed the importance of specific Ϫ35 recognition elements for the holoenzyme
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