Abstract
During transcription initiation by Escherichia coli RNA polymerase, a fraction of the homogeneous enzyme population has been kinetically shown to form two types of nonproductive complexes at some promoters: moribund complexes, which produce only abortive transcripts, and fully inactive ternary complexes (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457). Here we report biochemical isolation of the complexes arrested at the lambdaP(R) promoter and an analysis of their structure by DNA and protein footprintings. We found that the isolated promoter-arrested complexes retain a stoichiometric amount of sigma(70) subunit. Exonuclease III footprints of the arrested complexes are backtracked compared with that of the binary complex, and KMnO(4) footprinting reveals a decrease in the melting of DNA in the promoter region. Protein footprints of the retained sigma(70) have shown a more exposed conformation in region 3, compared with binary complexes. This feature is similar to that of the complexes arrested in inactive state during transcription elongation, indicating the existence of a common inactivating mechanism during transcription initiation and elongation. The possible involvement of the promoter arrest in transcriptional regulation is discussed.
Highlights
A plausible model of initiation at such promoters proposes two mutually exclusive pathways: a productive pathway leading to the synthesis of full-length transcripts and a dead-end pathway in which enzyme is likely to be arrested at these promoters [2,3,4,5]
Moribund complexes cannot escape from the abortive cycle to make full-length transcripts; rather, they slowly convert into the second type of arrested complexes
The synthesis of the full-length transcripts is completed within 5 min [2,3,4,5]
Summary
A plausible model of initiation at such promoters proposes two mutually exclusive pathways: a productive pathway leading to the synthesis of full-length transcripts and a dead-end pathway in which enzyme is likely to be arrested at these promoters [2,3,4,5]. After adding substrates containing the labeled initiating nucleotide, [␥-32P]GTP, this material was found to produce exclusively abortive transcripts, proving that the collected promoter DNA fragment contains no productive complexes.
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