Mechanism of transcript elongation control by RfaH

NusG/Spt 5蛋白是唯一已知的普遍保守的转录调节因子， 自最后一个普遍的共同祖先以来与RNA聚合酶共同进化。研究得最好的 这个家族的成员是大肠杆菌NusG，一种重要的管家蛋白， 与转录终止因子Rho一起沉默外源DNA， 转录，以及它的副产物RfaH，一种非必需的蛋白质，需要表达一些 水平获得的操纵子，在其前导区中包含ops序列。在我们 在之前的工作中，我们建立了RfaH作为模型来阐明转录机制 NusG蛋白赋予的持续合成能力。然而，我们和其他人最近的结构研究 提出NusG蛋白功能的范式转换模型。新的结构揭示了 意想不到的相互作用，并建议NusG蛋白可以促进延长， 促进易位，抑制回溯和旋转，陪伴新生RNA， 并限制柔性非模板DNA链。这些假设需要大量的 我们建议在目标1中使用RfaH进行验证。RfaH激活基因表达， 一部分是通过沉默Rho，Rho在RfaH控制的操纵子中施加强极性，但这些 操纵子缺乏典型的Ro识别序列。利用基因选择抑制基因， ΔrfaH，我们确定了Rho的柔性连接区的改变，我们建议破坏 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RNA下增强Rho作用。通过RfaH激活基因表达 被认为是由核糖体募集到缺失的RfaH靶RNA介导的 Shine-Dalgarno图案。在目标3中，我们将确定核糖体在哪里被募集到细胞中。 转录RNA聚合酶以及它是否在装载到mRNA上后扫描。我们还将测试 RfaH将转录和翻译偶联，并将寻找影响转录和翻译的新因素。 RfaH依赖性操纵子。

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.