Mechanistic studies of a new family of bacterial RNA chaperones

小非编码 RNA (sRNA) 调节几乎所有细菌物种的基因表达。 我的实验室表明，细菌质粒 F 家族中基因表达的调节是由一种称为 FinO 的 RNA 伴侣控制的，它促进反义 RNA 与其 mRNA 靶标的相互作用。在与 Xavier Charpentier（里昂）的一项新合作中，我们现已证明类似 FinO 的伴侣存在于多种细菌物种中，它们控制着不同的调节过程。 Charpentier 最近使用基因组方法鉴定了调节嗜肺军团菌 DNA 转导的 sRNA-mRNA 对，并证明嗜肺军团菌 FinO 是该调节网络所必需的。在此基础上，我们将致力于揭示嗜肺军团菌 FinO 调节 sRNA 功能的机制。我的实验室将利用我们在蛋白质-核酸相互作用的生物化学、生物物理和晶体学研究方面的专业知识来推导嗜肺军团菌 FinO 作用的结构机制。在前期工作中，我们已经证明嗜肺军团菌FinO特异性结合其sRNA，并且我们分离并结晶了嗜肺军团菌FinO的最小RNA结合结构域。我们假设嗜肺军团菌 FinO 通过与结合的 RNA 破坏发夹结构的稳定性来发挥作用，从而抑制 sRNA 和 mRNA 靶标之间的碱基配对。我们将使用一组生化测定来测试这一假设，并将通过晶体学和小角度 X 射线散射方法进一步探讨嗜肺军团菌 FinO 和 RNA 之间相互作用的结构原理。我们的机制模型将与 Charpentier 实验室合作，通过分析活细胞中结构引导的定点突变体的影响来进行测试。这将为比较不同细菌物种的 FinO 蛋白家族的功能奠定基础。该计划将为学生提供出色的培训机会，让他们在我的实验室内获得尖端结构生物学的经验，并有机会通过我们的国际合作将他们的培训扩展到微生物学和基因组学领域。**

Small non-coding RNAs (sRNAs) regulate gene expression in essentially all bacterial species. My laboratory showed that the regulation of gene expression in the F family of bacterial plasmids is controlled by an RNA chaperone, called FinO, that facilitates the interactions of antisense RNAs with their mRNA targets. In a new collaboration with Xavier Charpentier (Lyons), we have now shown that FinO-like chaperones exist in a variety of bacterial species where they control diverse regulatory processes. Charpentier has recently used genomic approaches to identify an sRNA-mRNA pair that regulates DNA transduction in L. pneumophila and he has shown that L. pneumophila FinO is required for this regulatory network. Building on this, we will work to uncover the mechanism by which L. pneumophila FinO regulates sRNA function. My lab will utilize our expertise in the biochemical, biophysical, and crystallographic study of protein-nucleic acid interactions to derive structural mechanisms for the action of L. pneumophila FinO. In preliminary work, we have already shown that L. pneumophila FinO specifically binds its sRNA and we have isolated and crystallized a minimal RNA-binding domain of L. pneumophila FinO. We hypothesize that L. pneumophila FinO acts by destabilizing hairpin structures with the bound RNA that inhibit base pairing between the sRNA and the mRNA target. We will test this hypothesis using a set of biochemical assays and will further probe the structural principles underlying the interactions between L. pneumophila FinO and RNA through crystallographic and small angle X-ray scattering approaches. Our mechanistic models will be tested through analysis of the effects of structure-guided site directed mutants in living cells in collaboration with the Charpentier lab. This will set the basis for the comparison of the function of the FinO family of proteins across diverse bacterial species. This program will offer outstanding training opportunities for students to gain experience in cutting edge structural biology within my lab, as well as an opportunity to extend their training to areas of microbiology and genomics through our international collaborations.**