TY - JOUR
T1 - Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase. In vitro transcription of photosynthesis and other genes
AU - Cullen, Paul J.
AU - Kaufman, Charles K.
AU - Bowman, William C.
AU - Kranz, Robert G.
PY - 1997
Y1 - 1997
N2 - To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the σ70 factor (R. capsulatus RNAP/σ70) was purified and characterized using two classical σ70 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 σ70 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/σ70 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/σ70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.
AB - To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the σ70 factor (R. capsulatus RNAP/σ70) was purified and characterized using two classical σ70 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 σ70 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/σ70 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/σ70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.
UR - http://www.scopus.com/inward/record.url?scp=0030876450&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.43.27266
DO - 10.1074/jbc.272.43.27266
M3 - Article
C2 - 9341173
AN - SCOPUS:0030876450
SN - 0021-9258
VL - 272
SP - 27266
EP - 27273
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -