Lactose promoter mutation P r115 activates an overlapping promoter within the lactose control region

Abstract

The Escherichia coli lac ‡ promoter mutation P r115, 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 S 1 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 P r115 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.