Investigations of Hfq-RNA Interactions

Hfq-RNA 相互作用的研究

• 批准号: 7097972
• 负责人: ANDREW L FEIG
• 金额: $ 23.62万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7097972
• 关键词: Clostridium perfringens; RNA binding protein; binding sites; crosslink; fluorescence resonance energy transfer; gel mobility shift assay; genetic library; intermolecular interaction; mass spectrometry; mutant; nucleotide analog; posttranslational modifications; protein binding; protein protein interaction; protein structure function; site directed mutagenesis; surface plasmon resonance

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.

描述(申请人提供)：Hfq是一种细菌类Sm蛋白，参与RNA生物化学的几个方面。这种蛋白质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靶标，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，但它似乎高保真地组装了正确的ncRNA-mRNA伙伴关系，而不会被困在不活跃的复合体中。这项提案中的工作试图从化学和生物的角度了解HfQ是如何发挥作用的。目的1研究链置换反应中涉及的分子握手，该反应改变了结构上被抑制的rpos mRNA的翻译状态。我们有证据表明，这种相互作用涉及三元络合物，并寻求捕获和分析反应路径上的中间体。然后，我们将基于体外数据，通过在体内评估这些突变的影响来测试我们模型的有效性。目的2更深入地探讨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干扰图谱和光交联研究，我们将表征高亲和力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产生的一系列Hfq突变体。利用这个突变体文库，我们已经开始区分与Hfq的各种生物学功能有关的接触面。作为体内这些研究的继续，我们将解决这些途径中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的问题。目标4将我们的工作带向一个新的方向。Hfq最近被认为与几种生物的细菌毒力有关。我们现在已经证明，来自完全性弧菌(一种导致气体坏疽等疾病的生物体)的一种名为VR-RNA的新型ncRNA以高亲和力结合Hfq。我们将绘制HfQ和VR-RNA是如何参与这种革兰氏阳性微生物毒力的图谱，并开始了解这些调控网络中的异同。