Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation

J Mol Biol. 1994 Jan 14;235(2):405-13. doi: 10.1006/jmbi.1994.1001.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / isolation & purification
  • DNA-Directed RNA Polymerases / physiology*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Genes, Bacterial / genetics
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics
  • Promoter Regions, Genetic / physiology*
  • Sigma Factor / chemistry
  • Sigma Factor / isolation & purification
  • Sigma Factor / physiology*
  • Transcriptional Activation / physiology*

Substances

  • Sigma Factor
  • RNA polymerase sigma 70
  • DNA-Directed RNA Polymerases