The ρ subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites. However, ρ is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of ρ. In order to study the position of ρ 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 (λ PR, λ PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the λ PR transcribed region) and PR/A1 (λ PR promoter with the T7 A1 transcribed region). Significant photoaffinity labeling of the ρ subunit was observed only on the templates containing the λ PR promoter region, regardless of the sequence of the transcribed region. These results indicate the molecular interactions responsible for the position of ρ 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 ρ subunit. These experiments corroborate the promoter dependence of ρ release. They also show a correlation between the release of ρ and stable binding of the transcript by the transcription complex.
|Original language||English (US)|
|Number of pages||8|
|State||Published - 1989|
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