Mechanistic studies of a new family of bacterial RNA chaperones

小的非编码RNA（sRNA）调节基本上所有细菌物种中的基因表达。 我的实验室表明，细菌质粒的F家族中的基因表达调控是由一种称为FinO的RNA伴侣控制的，它促进了反义RNA与其mRNA靶点的相互作用。在与Xavier Charpentier（里昂）的一项新合作中，我们现在已经证明FinO样分子伴侣存在于各种细菌物种中，它们控制着不同的调控过程。Charpentier最近使用基因组方法鉴定了调节L. pneumophila，他已经证明L. pneumophila FinO是该监管网络所必需的。在此基础上，我们将努力揭示L。嗜肺菌FinO调节sRNA功能。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、生物物理学和晶体学研究方面的专业知识，推导出L。嗜肺菌FinO.在初步工作中，我们已经证明了L。嗜肺军团菌FinO特异性结合其sRNA，我们已经分离并结晶了L.嗜肺菌FinO.我们假设L.嗜肺菌FinO通过使发夹结构与抑制sRNA和mRNA靶标之间碱基配对的结合RNA不稳定而起作用。我们将使用一系列生化分析来验证这一假设，并将进一步探索L。pneumophila FinO和RNA的分子结构。我们的机制模型将通过与Charpentier实验室合作分析活细胞中结构指导位点定向突变体的影响进行测试。这将为不同细菌物种中FinO蛋白家族功能的比较奠定基础。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前沿结构生物学的经验，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.**