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Volume 272, Number 43, Issue of October 24, 1997 pp. 27266-27273
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the Rhodobacter capsulatus Housekeeping RNA Polymerase
IN VITRO TRANSCRIPTION OF PHOTOSYNTHESIS AND OTHER GENES

(Received for publication, June 5, 1997, and in revised form, July 23, 1997)

From the Department of Biology, Washington University, St. Louis, Missouri 63130

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the ⁷⁰ factor (R. capsulatus RNAP/⁷⁰) was purified and characterized using two classical ⁷⁰ 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 ⁷⁰ 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/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/⁷⁰ enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.

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