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 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.
Cullen
P J
PJ
Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
Kaufman
C K
CK
Bowman
W C
WC
Kranz
R G
RG
D001426:Bacterial Proteins / Q000378:metabolism; D001429:Bacteriochlorophylls / Q000096:biosynthesis; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D016384:Consensus Sequence; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics; D045362:Cytochromes c2; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D047908:Intracellular Signaling Peptides and Proteins; D010788:Photosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011494:Protein Kinases; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012808:Sigma Factor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001429:Bacteriochlorophylls; D002352:Carrier Proteins; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012808:Sigma Factor; C028640:fructose-induced HPr-like protein, bacteria; D045362:Cytochromes c2; D011494:Protein Kinases; C488924:fruB protein, E coli; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases