Washburn R S, Jin D J, Stitt B L
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA.
J Mol Biol. 1996 Jul 19;260(3):347-58. doi: 10.1006/jmbi.1996.0405.
We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene. Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt'. The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified. We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE. The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation. We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants. NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG. However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE. These results suggest that NusG aids in the 3' end formation process. The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.
我们先前发现，大肠杆菌转录终止因子Rho中的nusD型突变增强了λcro基因内四个位点的体外转录终止。在此我们表明，早期终止位点是一个Rho依赖性终止位点tRE的一部分，其特性与先前鉴定的Rho依赖性位点λtR1和trp't相似。早期终止位点均为RNA聚合酶暂停位点，通过缺失分析和寡核苷酸阻断实验，确定了早期终止位点的一个共同的5' Rho进入位点（rutE）。我们表明，Rho026和Rho+都可以使用rutE作为终止的进入位点，但Rho026在tRE处释放新生RNA的效率更高。野生型和突变型Rhos的RNA依赖性ATP酶活性相似，它们结合游离RNA以及使用（rC）10寡聚物激活ATP酶的能力也相似。因此，我们认为在NusD Rho突变体中，定义RNA 3'末端形成位点的Rho-RNA聚合酶相互作用发生了改变。NusD Rho突变体对转录终止因子NusG的依赖性较小，但仍对其有反应。然而，在体外终止试验中添加NusG可使Rho+在tRE处更有效地终止。这些结果表明，NusG有助于3'末端形成过程。突变型Rhos在体外对NusG终止的依赖性降低，这为rho（nusD）细胞中λ生长较差提供了解释，因为它干扰了Rho依赖性位点处的λN介导的抗终止作用。
