Vieu Erwann, Rahmouni A Rachid
Centre de Biophysique Moléculaire, CNRS, rue Charles Sadron, 45071 Orléans, France.
J Mol Biol. 2004 Jun 18;339(5):1077-87. doi: 10.1016/j.jmb.2004.04.022.
Rho-dependent transcription termination at the phage lambda tR1 terminator is governed primarily by the upstream rut element that encodes two RNA regions rutA and rutB. The two regions are separated by the boxB RNA motif, which is believed to be dispensable for Rho activity but serves as a binding site for lambda N protein in the antitermination process. By using a minimal in vivo termination system, we show that the intervening boxB RNA motif has a double function in the mechanisms of termination/antitermination at lambdatR1. As a folded hairpin structure, it acts as a clamp that holds rutA and rutB side by side for optimal interactions with Rho leading to efficient termination. Conversely, the binding of N protein to boxB induces antitermination at lambdatR1 by preventing access of Rho to the rut sequences. This dual role was clearly shown in vivo by studying the effects of multiple mutations within the boxB hairpin stem on transcription termination and by substituting the N/boxB couple with the unrelated coat protein of phage MS2 and its stem-loop RNA binding site.
噬菌体λ tR1终止子处的Rho依赖性转录终止主要由上游的rut元件控制,该元件编码两个RNA区域rutA和rutB。这两个区域被boxB RNA基序隔开,据信该基序对Rho活性并非必需,但在抗终止过程中作为λ N蛋白的结合位点。通过使用一个最小的体内终止系统,我们表明,居间的boxB RNA基序在λ tR1的终止/抗终止机制中具有双重功能。作为一种折叠的发夹结构,它充当一个夹子,将rutA和rutB并排固定,以便与Rho进行最佳相互作用,从而实现高效终止。相反,N蛋白与boxB的结合通过阻止Rho接近rut序列而在λ tR1处诱导抗终止。通过研究boxB发夹茎内多个突变对转录终止的影响,以及用噬菌体MS2的无关外壳蛋白及其茎环RNA结合位点替代N/boxB对,这种双重作用在体内得到了明确展示。