Upstream interactions at the lambda p(RM) promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter

The rightward regulatory region of bacteriophage lambda contains two promoters, p(RM) and p(R), which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of p(RM)'s ATR. We determined the effects on RNA polymerase-p(RM) promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the α subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the α subunit on open complex formation at p(RM). However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at p(RM) stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of p(R) by upstream interactions. The possible involvement of the α subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of p(RM)-bound RNA polymerase with open complex formation at p(RM) is discussed.