UPSTREAM INTERACTIONS AT THE LAMBDA-P(RM) PROMOTER ARE SEQUENCE NONSPECIFIC AND ACTIVATE THE PROMOTER TO A LESSER EXTENT THAN AN INTRODUCEDUP ELEMENT OF AN RIBOSOMAL-RNA PROMOTER

The rightward regulatory region of bacteriophage lambda contains two p romoters, p(RM) and p(R), which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two p romoters has an upstream (A+T)-rich region (ATR) within the sequence f rom -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 alph a subunit, known to affect the upstream interaction, We find small (tw o- to threefold) effects of selected mutations in the or subunit on op en complex formation at p(RM). However, the (presumably upstream) inte ractions underlying these effects are sequence nonspecific, as they ar e 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 bin ding event itself, Less extensive studies Likewise provide evidence fo r a two- to threefold activation of p(R) by upstream interactions. The possible involvement of the alpha subunit in the previously character ized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 27 0:30428-30433, 1995) interference of p(R)-bound RNA polymerase with op en complex formation at p(RM) is discussed.