The Escherichia coli lac‡ promoter mutation Pr115, an A · T to T · A transversion at +1 (the transcription initiation site of the lac wild-type and lac UV5 promoters), creates a new "-10 region"-like sequence starting at +1. We show that this mutation activates a new RNA polymerase binding site (P115) that overlaps with, and is shifted 12 base-pairs downstream from, the wild-type RNA polymerase binding site (P1). Nuclease S1 mapping studies and RNA polymerase protection experiments in vitro indicate that, in the absence of CAP-cAMP, this new site is used preferentially over the P1 site. In vivo, β-galactosidase assays of the Pr115 mutation in combination with mutations of the P1 "-35 region" demonstrate that the P1 -35 region sequences are not involved in the interaction between RNA polymerase and P115 in the absence of CAP-cAMP; therefore P115 is an independent binding site. The presence of CAP-cAMP in vivo stimulates polymerase binding and initiation at P1, which serves to block polymerase from binding at P115.
|Number of pages||9|
|Journal||Journal of Molecular Biology|
|State||Published - Oct 5 1985|
Bibliographical noteFunding Information:
The authors thank Yu Xian-Ming. Paul Lambert and Marvin Wickens for critical reading of this manuscrupt. This work was supported by the National Institute of Health grant GM19670 and a 3M Foundation grant to W.S.R. and M.L.P. was supported in part by the National Institute of Health training grant GM07215-07.
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology