title: Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation.
abstract: The role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated. For this purpose, we constructed a nested set of E. coli rpoD deletion mutants generating carboxy-terminally truncated sigma 70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant sigma 70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of sigma 70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect "extended minus 10" promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region. However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences. These results establish that -35 DNA recognition by sigma 70 is not essential for these cases. Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region. Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the sigma 70 subunit, within a "contact site II", which extends at least from conserved region 3.2 to the upstream end of region 4.2. Moreover, as in the case of contact site I on the alpha subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites.
authors.affiliation: Institute of Cell and Molecular Biology, University of Edinburgh, Scotland, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: B
authors.initials: B
authors.lastname: Grimes
authors.forename: N
authors.initials: N
authors.lastname: Fujita
authors.forename: K
authors.initials: K
authors.lastname: Makino
authors.forename: R A
authors.initials: RA
authors.lastname: Malloch
authors.forename: R S
authors.initials: RS
authors.lastname: Hayward
authors.forename: A
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D012808:Sigma Factor / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases