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J. Biol. Chem., Vol. 278, Issue 14, 12344-12355, April 4, 2003
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From the The study of mutant enzymes can reveal important
details about the fundamental mechanism and regulation of RNA
polymerase, the central enzyme of gene expression. However, such
studies are complicated by the multisubunit structure of RNA polymerase
and by its indispensability for cell growth. Previously, mutant RNA polymerases have been produced by in vitro assembly from
isolated subunits or by in vivo assembly upon
overexpression of a single mutant subunit. Both approaches can fail if
the mutant subunit is toxic or incorrectly folded. Here we describe an
alternative strategy, co-overexpression and in vivo
assembly of RNA polymerase subunits, and apply this method to
characterize the role of sequence insertions present in the
Escherichia coli enzyme. We find that co-overexpression of
its subunits allows assembly of an RNA polymerase lacking a 188-amino
acid insertion in the
Co-overexpression of Escherichia coli
RNA Polymerase Subunits Allows Isolation and Analysis of Mutant Enzymes
Lacking Lineage-specific Sequence Insertions*
,§,
**
Department of Microbiology, Ohio State
University, Columbus, Ohio 43210, the ¶ Laboratory of
Molecular Biophysics, Rockefeller University, New York, New York 10021, and the Department of Bacteriology, University of Wisconsin,
Madison, Wisconsin 53706
' subunit. Based on experiments with this and
other mutant E. coli enzymes with precisely excised
sequence insertions, we report that the ' sequence insertion and, to
a lesser extent, an N-terminal sequence insertion confer characteristic stability to the open initiation complex, frequency of
abortive initiation, and pausing during transcript elongation relative
to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.
*
This work was supported by National Institutes of Health
Grant GM38660 and Department of Agriculture Grant WIS04022 (to R. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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