A cryptic promoter in the O(R) region of bacteriophage lambda

Abstract

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

Interference of PR-bound RNA Polymerase with Open Complex Formation at PRM Is Relieved by a 10-Base Pair Deletion between the Two Promoters

“…The times of 5 and 60 min refer to the duration of the incubation of DNA and RNA polymerase prior to initiation of RNA synthesis. In the lanes labeled P R Ϫ , note the appearance of a larger transcript, which likely originated at the P␣ promoter described by Gussin and co-workers (22). two-polymerase complexes (i.e.…”

“…We (1, 2) as well as Gussin and co-workers (3,4) have shown that the presence of the P R promoter has a negative effect on the ability of RNA polymerase to form open complexes at P RM . The use of P R mutants allowed Gussin's group to demonstrate that conversely, the presence of an RNA polymerase at P RM negatively affects the utilization of a (weakened) P R promoter as well (5).…”

“…7. In order to conform to the kinetic results reviewed in the introduction (1,3,4), no interference would occur in the intermediate (closed) complex. There is indirect evidence (7) for the occurrence of a conformational change in RNA polymerase in the process of formation of an open promoter complex after the initial contact between the enzyme and promoter DNA.…”

“…Thus nearly every RNA polymerase binding at P RM does so in the context of another polymerase situated at P R . Our own work (1, 2) as well as more extensive kinetic analysis by Gussin and his group (3,4) has demonstrated that the interference with complex formation at P RM manifests itself not at the initial bimolecular (binding) step but rather in some subsequent step involving conformational transitions in RNA polymerase and/or DNA. The interference slows down but does not prevent open complex formation at P RM ; ultimately open complexes at both promoters do co-exist on the same DNA fragment (Refs.…”

“…Reduction of the distance between P R and P RM by one base pair further slows open complex formation at P RM (4). The effects of considerably shorter separation between the P R and P RM promoters can be assessed from studies on other lambdoid phages.…”