Zhou Ying, Shi Ting, Mozola Mark A, Olson Eric R, Henthorn Karla, Brown Susan, Gussin Gary N, Friedman David I
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.
J Bacteriol. 2006 Mar;188(6):2222-32. doi: 10.1128/JB.188.6.2222-2232.2006.
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.
噬菌体λ的N蛋白与大肠杆菌的Nus蛋白在RNA位点NUT处相互作用，将RNA聚合酶（RNAP）修饰成一种能够越过转录终止子的形式。这些相互作用被认为是N介导的抗终止作用有效性的主要决定因素。我们提供的证据表明，相关的启动子，在这种情况下是λ早期P（R）启动子，即使修饰发生在中间cro基因下游的位点（NUTR），也能影响N介导的RNAP修饰。正如遗传分析所预测并经体内转录研究证实的那样，P（R）中-14和-45位的两个突变组合（产生P（R-GA））会降低N修饰的有效性，而-30位的另一个突变（产生P（R-GCA））会抑制这种效应。在体内，P（R-GA）指导的转录水平是野生型水平的两倍，而P（R-GCA）指导的转录与野生型启动子指导的转录相同。然而，体外P（R-GA）处开放复合物形成的速率大约是野生型P（R）的三分之一。我们将这种明显的差异归因于P（R-GCA）中的突变对启动子清除的影响。基于体内实验，对我们结果的一个合理的解释是，转录增加可能导致无法与NUT RNA形成活性抗终止复合物，进而导致无法通读下游终止位点。通过阻断抗终止作用从而抑制晚期功能的表达，通过nut位点增加转录的效应在噬菌体发育早期维持基因表达的适当调控方面可能具有重要的生理意义。
