title: On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter.
abstract: Bacterial promoters of the extended -10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended -10 Escherichia coli galP1 promoter at temperatures as low as 6 degrees C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended -10 motif and/or containing the -35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif. However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent. Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif. We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations.
authors.affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006493:Heparin / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011196:Potassium Permanganate / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry*; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014764:Viral Proteins; D011196:Potassium Permanganate; D006493:Heparin; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I