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(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
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
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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