Investigations of Hfq-RNA Interactions

描述（由申请人提供）：Hfq是一种细菌sm样蛋白，参与RNA生物化学的几个方面。该蛋白在转录后基因调控过程中促进RNA-RNA相互作用。在这些途径中，小的非编码rna （ncRNAs）调节mRNA靶点以促进对冷休克、热休克、渗透休克和氧化应激等环境条件的适应。Hfq与许多RNA伙伴紧密相互作用，但它似乎以高保真度组装正确的ncRNA-mRNA伙伴关系，而不会被困在非活性复合物中。本提案的工作旨在从化学和生物学的角度理解Hfq的功能。目的1着眼于参与链位移反应的分子握手，该反应改变了结构上受抑制的rpoS mRNA的翻译状态。我们有证据表明这种相互作用涉及三元配合物，并试图沿着反应途径捕获和分析中间体。然后，我们将通过评估这些突变在体内的影响，来测试基于体外数据的模型的有效性。目的2更深入地探讨了Hfq对其ncRNA和mRNA伴侣的识别所涉及的二元相互作用。通过使用核苷酸类似物干涉作图和光交联研究，我们将描述高亲和结合所需的结构特征和原子接触。Aim 3进一步分析了我们之前生成的一系列Hfq突变体。利用这个突变体库，我们已经开始区分涉及Hfq各种生物学功能的接触面。作为这些体内研究的延续，我们将解决是否有额外的蛋白质成分作为更大的大分子组合的一部分参与这些途径的问题。目标4将我们的工作带入了一个新的方向。Hfq最近与几种生物的细菌毒力有关。我们现在已经证明，来自C. perfingens（一种引起气性坏疽和其他疾病的生物体）的一种名为VR-RNA的新型ncRNA高亲和力地结合Hfq。我们将绘制Hfq和VR-RNA如何参与这种革兰氏阳性生物体的毒力，并开始了解这些调节网络的异同。

DESCRIPTION (provided by applicant): Hfq is a bacterial Sm-like protein involved in several aspects of RNA biochemistry. This protein facilitates RNA-RNA interactions during post-transcriptional gene regulation. In these pathways, small non-coding RNAs (ncRNAs) regulate mRNA targets to facilitate adaption to environmental conditions like cold shock, heat shock, osmotic shock and oxidative stress. Hfq interacts tightly with many RNA partners and yet it seems to assemble the correct ncRNA-mRNA partnerships with high fidelity without becoming trapped in inactive complexes. The work in this proposal seeks to understand from a chemical and biological perspective how Hfq functions. Aim 1 looks at the molecular handshakes involved in the strand displacement reaction which alters the translational state of the structurally repressed rpoS mRNA. We have evidence that this interaction involves ternary complexes and seek to trap and analyze intermediates along the reaction pathway. We will then test the validity of our models based on in vitro data by assessing in vivo, the effect of these mutations. Aim 2 probes more deeply the binary interactions invovled in Hfq's recognition of its ncRNA and mRNA partners. By using nucleotide analog interferences mapping and photocrosslinking studies we will characterize the structural features and atomic contacts required for high affinity binding. Aim 3 further analyzes a series of Hfq mutants that we have previously generated. Using this library of mutants, we have begun to differentiate the contact surfaces involved in Hfq's various biological functions. As a continuation of these studies in vivo, we will be addressing the question of whether additional protein components are involved in some of these pathways as part of a larger macromolecular assemblages. Aim 4 takes our work in a new direction. Hfq has recently been implicated in bacterial virulence in several organisms. We have now shown that a novel ncRNA called VR-RNA from C. perfingens (an organism that causes gas gangrene among other ailments) binds Hfq with high affinity. We will be map how Hfq and VR-RNA are invovled in virulence of this Gram positive organism and begin to understand the similarities and differences in these regulatory networks.