Release of the sigma subunit from Escherichia coli RNA polymerase transcription complexes is dependent on the promoter sequence

Abstract

The sigma subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites. However, sigma is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of sigma. In order to study the position of sigma release, we have developed a method to quantify the photoaffinity labeling produced by an aryl azide positioned at the leading (5'-) end of nascent RNA, as a function of the transcript length [Stackhouse, T.M., & Meares, C.F. (1988) Biochemistry 27, 3038-3045]. Here we compare photoaffinity labeling of transcription complexes containing three natural bacteriophage promoters (lambda PR, lambda PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the lambda PR transcribed region) and PR/A1 (lambda PR promoter with the T7 A1 transcribed region). Significant photoaffinity labeling of the sigma subunit was observed only on the templates containing the lambda PR promoter region, regardless of the sequence of the transcribed region. These results indicate the molecular interactions responsible for the position of sigma release from the transcription complex mainly involve the nucleotide sequence of the promoter region--rather than the transcribed region--of the DNA template. Further studies on transcription complexes containing the A1/PR and the PR/A1 templates were performed, using polyclonal antibodies against the holoenzyme or against the sigma subunit. These experiments corroborate the promoter dependence of sigma release. They also show a correlation between the release of sigma and stable binding of the transcript by the transcription complex.