Mechanism of transcript elongation control by RfaH

NusG/Spt5蛋白是唯一已知的普遍保守的转录调节因子 自最后一个全球共同祖先以来，与RNA聚合酶共同进化。研究最多的人 这个家族的成员是大肠杆菌NusG，它是一种重要的管家蛋白质，具有 与转录终止因子Rho一起沉默外源DNA和反义 转录，及其伴随的RfaH，一种非必需的蛋白质，表达几个 水平获取的操纵子，在其前导区域中包含操作序列。在我们的 在之前的工作中，我们建立了RfaH作为模型来解释转录机制 NusG蛋白赋予的加工能力。然而，我们和其他人最近的结构研究 提出NusG蛋白质如何发挥作用的范式转换模型。新的结构揭示了 意想不到的相互作用，并表明NusG蛋白可能通过 促进转位，抑制回溯和旋转，陪伴新生的RNA， 并对柔性非模板DNA链进行限制。这些假设需要广泛的 我们在AIM 1中建议用RfaH进行的验证。RfaH激活了 部分是通过使Rho沉默，这在RfaH控制的操纵子中施加了强极性，然而这些 操纵子缺乏标准的Ro识别序列。利用基因选择抑制基因的表达 ΔrfaH，我们发现了Rho的一个灵活连接区的改变，我们认为这种改变会扰乱 终止所需的变构信号。在目标2中，我们将研究连接区的作用 和NusG在次优RNAs中增强Rho作用。RfaH对基因表达的激活作用 被认为是通过向缺失的RfaH靶RNA募集核糖体来介导的 闪亮的达尔加诺图案。在目标3中，我们将确定核糖体被招募到哪里 转录RNA聚合酶，以及它在加载mRNA后是否进行扫描。我们还将测试是否 RfaH将转录和翻译结合在一起，将寻找影响翻译的新因素 依赖于RfaH的操纵子。

NusG/Spt5 proteins are the only known universally conserved transcriptional regulators that coevolved with RNA polymerase since the last universal common ancestor. The best studied members of this family are Escherichia coli NusG, an essential housekeeping protein that acts together with the transcription termination factor Rho to silence foreign DNA and antisense transcription, and its paralog RfaH, a non-essential protein required for expression of a few horizontally-acquired operons that contain an ops sequence in their leader regions. In our previous work, we established RfaH as a model to elucidate the mechanism of transcription processivity conferred by NusG proteins. However, recent structural studies by us and others propose paradigm-shifting models for how NusG proteins function. The new structures revealed unexpected interactions and suggested that NusG proteins may promote elongation by facilitating translocation, inhibiting backtracking and swiveling, chaperoning the nascent RNA, and constraining the flexible nontemplate DNA strand. These hypotheses require extensive validation that we propose to carry out with RfaH in Aim 1. RfaH activates gene expression in part by silencing Rho, which imposes strong polarity in RfaH-controlled operons, yet these operons lack canonical Ro recognition sequences. Using a genetic selection for suppressors of ΔrfaH, we identified alterations in a flexible connector region of Rho that we propose disrupt an allosteric signal required for termination. In Aim 2, we will study the role of the connector region and NusG in potentiating Rho action at suboptimal RNAs. Activation of gene expression by RfaH is thought to be mediated by recruitment of ribosome to RfaH-target RNAs that are missing Shine-Dalgarno motifs. In Aim 3, we will determine where the ribosome is recruited to the transcribing RNA polymerase and whether it scans after loading on mRNA. We will also test if RfaH couples transcription and translation and will look for new factors affecting translation of RfaH-dependent operons.