Role of the sigma70 subunit of Escherichia coli RNA polymerase in transcription activation

Ashok Kumar, Brenda Grimes, Nobuyuki Fujita, Kozo Makino, Richard A. Malloch, Richard S. Hayward, Akira Ishihama

Research output: Contribution to journalArticle

86 Scopus citations

Abstract

The role of the σ70 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 σ70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant σ70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of σ70 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 σ70 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 σ70 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 α 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.

Original languageEnglish (US)
Pages (from-to)405-413
Number of pages9
JournalJournal of molecular biology
Volume235
Issue number2
DOIs
StatePublished - Jan 1994
Externally publishedYes

Keywords

  • Protein-protein contact
  • RNA polymerase
  • Sigma subunit
  • Transcription factor
  • Transcription regulation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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