Evidence that the promoter can influence assembly of antitermination complexes at downstream RNA sites

The N protein of phage lambda acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the lambda early P(R) promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P(R), at positions -14 and -45 (yielding P(R-GA)), reduces effectiveness of N modification, while an additional mutation at position -30 (yielding P(R-GCA)) suppresses this effect. In vivo, the level of P(R-GA)-directed transcription was twice as great as the wild-type level, while transcription directed by P(R-GCA) was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P(R-GA) in vitro was roughly one-third the rate for wild-type P(R). We ascribe this apparent discrepancy to an effect of the mutations in P(R-GCA) on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.