Mechanism of transcript elongation control by RfaH

描述（由申请人提供）：在生命的所有领域中，长链RNA的有效合成需要辅助蛋白将RNA聚合酶修饰为高度加工状态。细菌转录因子RfaH属于普遍保守的NusG蛋白家族，激活水平转移操纵子的表达，编码细胞壁和胶囊成分、抗生素和毒力因子，所有这些都受rho介导的强极性影响。RfaH的作用依赖于一种称为ops的DNA序列，该序列在延伸期间介导RfaH募集到RNA聚合酶。我们详细分析了RfaH对转录的影响，并确定了其与RNA聚合酶和ops DNA相互作用的决定因素。这些研究使我们能够提出RfaH与转录延伸复合体相互作用的模型，并表明RfaH作为一个加工钳，稳定RNA聚合酶与DNA和RNA的接触。尽管其他研究小组的研究证实，这种机制似乎是古老而普遍的，但我们的研究结果表明，它对RfaH基因表达的100倍激活的作用相对较小。RfaH的主要作用似乎依赖于通过与Rho辅因子NusG竞争并将核糖体招募到新生mRNA来阻断Rho依赖性终止。第二种作用模式是由RfaH和核糖体蛋白S10之间的相互作用介导的，这需要RfaH的c端结构域从α-螺旋发夹完全重新折叠成ß-桶。这种变形与在感染性朊病毒形成过程中发生的变形一样引人注目。在这个建议中，我们将使用生化，遗传和结构的组合

DESCRIPTION (provided by applicant): In all domains of life, efficient synthesis of long RNAs requires accessory proteins that modify RNA polymerase into a highly processive state. Bacterial transcription factor RfaH, which belongs to a universally conserved family of NusG proteins, activates expression of horizontally-transferred operons encoding cell wall and capsule components, antibiotics, and virulence factors, all of which are subject to strong Rho-mediated polarity. RfaH action depends on a DNA sequence called ops that mediates RfaH recruitment to RNA polymerase during elongation. We carried out detailed analysis of RfaH effects on transcription and identified determinants for its interactions with RNA polymerase and the ops DNA. These studies allowed us to propose a model for RfaH interactions with the transcription elongation complex and suggested that RfaH acts as a processivity clamp which stabilizes RNA polymerase contacts with DNA and RNA. Although studies in other groups confirmed that this mechanism appears to be ancient and ubiquitous, our results revealed that it makes a relatively small contribution to a dramatic, hundred-fold activation of gene expression by RfaH. The main effect of RfaH appears to rely on blocking Rho-dependent termination by competing with a Rho cofactor NusG and recruiting the ribosome to the nascent mRNA. The second mode of action is mediated by interactions between RfaH and ribosomal protein S10, which require a complete refolding of the C-terminal domain of RfaH from an α-helical hairpin into ß-barrel. This metamorphosis is as dramatic as the one proposed to occur during formation of an infectious form of prions. In this proposal, we will use a combination of biochemical, genetic, and structural approaches to pursue these unexpected findings. First, we will study conformational transitions of RfaH C-terminal domain during its life cycle, from synthesis on the ribosome to hypothetical recycling upon dissociation from RNA polymerase at the end of an operon. Second, we will follow the events that trigger RfaH domain dissociation, a prelude to refolding, during recruitment at the ops site. Third, we will begin to elucidate the mechanism by which RfaH activates translation of mRNAs that lack functional Shine- Dalgarno elements.