Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.
- 23 May 1995
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 92 (11) , 4902-4906
- https://doi.org/10.1073/pnas.92.11.4902
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.Keywords
This publication has 32 references indexed in Scilit:
- Location, structure, and function of the target of a transcriptional activator protein.Genes & Development, 1994
- Domain organization of RNA polymerase α subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA bindingCell, 1994
- Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymeraseMolecular Microbiology, 1992
- Bipartite functional map of the E. coli RNA polymerase α subunit: Involvement of the C-terminal region in transcription activation by cAMP-CRPCell, 1991
- [6] Efficient site-directed mutagenesis using uracil-containing DNAPublished by Elsevier ,1991
- Blocking of the Initiation-to-Elongation Transition by a Transdominant RNA Polymerase MutationScience, 1990
- Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoterJournal of Molecular Biology, 1984
- Subunits of RNA polymerase in function and structureJournal of Molecular Biology, 1976
- Subunits of RNA polymerase in function and structure: V. Maturation in vitro of core enzyme from Escherichia coliJournal of Molecular Biology, 1974
- Subunits of RNA polymerase in function and structure: IV. Enhancing role of sigma in the subunit assembly of Escherichia coli RNA polymeraseJournal of Molecular Biology, 1973