title: Oligomerization of the E. coli core RNA polymerase: formation of (α2ββ'ω)2-DNA complexes and regulation of the oligomerization by auxiliary subunits.
abstract: In this work, using multiple, dissimilar physico-chemical techniques, we demonstrate that the Escherichia coli RNA polymerase core enzyme obtained through a classic purification procedure forms stable (α(2)ββ'ω)(2) complexes in the presence or absence of short DNA probes. Multiple control experiments indicate that this self-association is unlikely to be mediated by RNA polymerase-associated non-protein molecules. We show that the formation of (α(2)ββ'ω)(2) complexes is subject to regulation by known RNA polymerase interactors, such as the auxiliary SWI/SNF subunit of RNA polymerase RapA, as well as NusA and σ(70). We also demonstrate that the separation of the core RNA polymerase and RNA polymerase holoenzyme species during Mono Q chromatography is likely due to oligomerization of the core enzyme. We have analyzed the oligomeric state of the polymerase in the presence or absence of DNA, an aspect that was missing from previous studies. Importantly, our work demonstrates that RNA polymerase oligomerization is compatible with DNA binding. Through in vitro transcription and in vivo experiments (utilizing a RapA(R599/Q602) mutant lacking transcription-stimulatory function), we demonstrate that the formation of tandem (α(2)ββ'ω)(2)-DNA complexes is likely functionally significant and beneficial for the transcriptional activity of the polymerase. Taken together, our findings suggest a novel structural aspect of the E. coli elongation complex. We hypothesize that transcription by tandem RNA polymerase complexes initiated at hypothetical bidirectional "origins of transcription" may explain recurring switches of the direction of transcription in bacterial genomes.
authors.affiliation: Department of Chemistry and Biochemistry, Lamar University, Beaumont, Texas, United States of America.
authors.forename: Seema G
authors.initials: SG
authors.lastname: Kansara
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Sukhodolets
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases