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Volume 272, Number 34,
Issue of August 22, 1997
pp. 21558-21564
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Characterization, and Reconstitution of
DNA-dependent RNA Polymerases from Caulobacter
crescentus
(Received for publication, March 17, 1997, and in revised form, June 4, 1997)
Jianguo
Wu
,
Noriko
Ohta
,
Andrew K.
Benson
,
Alexander J.
Ninfa
and
Austin
Newton
From the Department of Molecular Biology, Princeton University,
Princeton, New Jersey 08544
Cell differentiation in the Caulobacter
crescentus cell cycle requires differential gene expression that
is regulated primarily at the transcriptional level. Until now,
however, a defined in vitro transcription system for the
biochemical study of developmentally regulated transcription factors
had not been available in this bacterium. We report here the
purification of C. crescentus RNA polymerase holoenzymes
and resolution of the core RNA polymerase from holoenzymes by
chromatography on single-stranded DNA cellulose. The three RNA
polymerase holoenzymes E 54, E 32, and
E 73 were reconstituted exclusively from purified
C. crescentus core and sigma factors. Reconstituted
E 54 initiated transcription from the
54-dependent fljK promoter of
C. crescentus in the presence of the transcription
activator FlbD, and active E 32 specifically initiated
transcription from the 32-dependent promoter
of the C. crescentus heat-shock gene dnaK. For
reconstitution of the E 73 holoenzyme, we overexpressed
the C. crescentus rpoD gene in Escherichia coli
and purified the full-length 73 protein. The
reconstituted E 73 recognized the
70-dependent promoters of the E. coli
lacUV5 and neo genes, as well as the
73-dependent housekeeping promoters of the
C. crescentus pleC and rsaA genes. The ability
of the C. crescentus E 73 RNA polymerase to
recognize E. coli
70-dependent promoters is consistent
with relaxed promoter specificity of this holoenzyme previously
observed in vivo.

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Copyright © 1997 by the American Society for Biochemistry and Molecular Biology.
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