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RNA的有效合成需要辅助蛋白，其将RNA聚合酶修饰成高度加工状态。细菌转录因子RfaH属于普遍保守的NusG蛋白家族，激活编码细胞壁和荚膜组分、抗生素和毒力因子的水平转移操纵子的表达，所有这些都受到强Rho介导的极性的影响。RfaH的作用依赖于一个称为ops的DNA序列，该序列在延伸过程中介导RfaH向RNA聚合酶的募集。我们对RfaH对转录的影响进行了详细的分析，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与转录延伸复合物相互作用的模型，并表明RfaH充当稳定RNA聚合酶与DNA和RNA接触的持续合成钳。虽然其他研究小组的研究证实，这种机制似乎是古老和普遍存在的，我们的研究结果表明，它作出了相对较小的贡献，一个戏剧性的，百倍的基因表达激活RfaH。RfaH的主要作用似乎依赖于通过与Rho辅因子NusG竞争并将核糖体募集到新生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末端结构域从？-螺旋发夹变成？每桶这种变形与朊病毒传染性形成过程中发生的变形一样引人注目。在这个建议中，我们将使用生物化学，遗传学和结构学相结合的方法。 探索这些意想不到的发现。首先，我们将研究RfaH C-末端结构域在其生命周期中的构象转变，从核糖体上的合成到操纵子末端RNA聚合酶解离后的假设回收。第二，我们将遵循触发RfaH结构域解离的事件，这是在ops位点募集期间重折叠的前奏。第三，我们将开始阐明RfaH激活缺乏功能性Shine-Dalgarno元件的mRNA翻译的机制。

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 ?-helical hairpin into ?-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.