PMID:8289270
| Citation |
Kumar, A, Grimes, B, Fujita, N, Makino, K, Malloch, RA, Hayward, RS and Ishihama, A (1994) Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation. J. Mol. Biol. 235:405-13 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1006/jmbi.1994.1001 |
| Keywords |
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 |
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