Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions.
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 beta' subunit. Based on experiments with this and other mutant E. coli enzymes with precisely excised sequence insertions, we report that the beta' sequence insertion and, to a lesser extent, an N-terminal beta 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.
Artsimovitch
Irina
I
Department of Microbiology, Ohio State University, Columbus, Ohio 43210, USA.
Svetlov
Vladimir
V
Murakami
Katsuhiko S
KS
Landick
Robert
R
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D066298:In Vitro Techniques; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary
D004251:DNA Transposable Elements; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; C105078:RNA polymerase beta subunit