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PMID: 7761421 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 92 ·No. 11 ·1995-05-23 ·Pages 4902-6

Tang H, Severinov K, Goldfarb A, Ebright RH

Abstract

We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.

MeSH Terms
Base Sequence Chromatography, Affinity DNA/metabolism DNA-Directed RNA Polymerases/biosynthesis,genetics,isolation & purification Escherichia coli/enzymology,genetics Macromolecular Substances Molecular Sequence Data Oligodeoxyribonucleotides Plasmids Promoter Regions, Genetic Recombinant Proteins/biosynthesis,isolation & purification Sequence Tagged Sites Time Factors Transcription, Genetic
Chemicals
Macromolecular Substances Oligodeoxyribonucleotides Recombinant Proteins DNA DNA-Directed RNA Polymerases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Tang H
Department of Chemistry, Rutgers University, New Brunswick, NJ 08855, USA.
Severinov K
Goldfarb A
Ebright R H
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32 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.92.11.4902
Published
1995-05-23
Pages
4902-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC41815
Subset
IM
Grants
NIGMS NIH HHS · GM30717 · United States
NIGMS NIH HHS · GM41376 · United States
NIGMS NIH HHS · GM51527 · United States
External Links
PubMed source DOI: 10.1073/pnas.92.11.4902 PMC Full Text View journal details
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