Control of bacterial gene expression by the FinO family of RNA chaperones

背景资料：小的非编码RNA（sRNA）在称为RNA伴侣的蛋白质的帮助下调节基本上所有细菌物种中的基因表达。RNA分子伴侣的FinO家族是最重要和最广泛的分子伴侣家族之一，其在细菌应激反应和水平基因转移（HGT）的调节中起作用。我们的合作者Xavier Charpentier（里昂）在军团菌中发现了一种新的FinO伴侣，称为RocC。他的研究小组表明，RocC结合了一种称为RocR的单一sRNA，从而稳定了RocR，并促进了控制HGT的特定mRNA靶点的抑制。 我们开始了解RocC如何特异性结合RNA以及它如何促进sRNA-mRNA配对。我们发现RocC FinO结构域结合RocR的发夹尾转录终止子，并且我们继续确定RocC FinO结构域与RocR终止子复合的晶体结构。这种结构提供了任何FinO分子伴侣与其RNA结合的第一个视图，并为我们的关键假设提供了基础，这些假设将指导我们在本计划的下一个学期的实验。 假设：我们提出FinO分子伴侣以读取3' ssRNA尾的长度的方式结合转录终止子结构。此外，我们提出，结合机制允许sRNA的重塑，使sRNA-mRNA配对。 具体目标1。完善我们对RocC-RocR相互作用的理解。 为了了解我们的RocC-RocR结构的功能含义，我们正在RocC中构建大量定点点突变以及RocR发夹尾中的修饰，以了解结合亲和力/特异性的关键相互作用。这些突变的影响将通过生物化学和生物物理测定进行探测，并将在军团菌中评估其对RocR稳定性、基因表达和能力的影响。具体目标2。探索RocC结合对RocR结构和与mRNA靶标相互作用的影响。 在初步工作中，我们发现RocR在终止子发夹和sRNA“种子”序列之间采用假结结构，我们提出这必须展开以促进sRNA-mRNA配对。我们将通过足迹法、NMR研究、假结结构的结晶和通过小角X射线散射分析更大规模的RocC-RocR结构来测试这一想法。这些假设将通过军团菌体内研究进一步检验。具体目标3。了解FinO分子伴侣在细菌中的广泛功能。使用一组跨越FinO系统发育树的FinO分子伴侣，我们将比较它们的结构，RNA结合特异性和RNA配对活性，以了解FinO功能的保守程度，这些作用将在结构域交换实验中进一步测试。这些实验应该揭示这些分子伴侣之间特异性差异的基础，以及这些蛋白质如何促进RNA-RNA配对。

Background: Small non-coding RNAs (sRNAs) regulate gene expression in essentially all bacterial species with the assistance of proteins called RNA chaperones. The FinO family of RNA chaperones is one of the most important and widespread chaperone families with roles in regulation of bacterial stress responses and horizontal gene transfer (HGT). Our collaborator, Xavier Charpentier (Lyon) discovered a new FinO chaperone in Legionella called RocC. His group showed that RocC binds a single sRNA called RocR, thereby stabilizing RocR and facilitating the repression of specific mRNA targets that control HGT. We set out to understand how RocC specifically binds RNA and how it facilitates sRNA-mRNA pairing. We showed that the RocC FinO domain binds the hairpin-tail transcription terminator of RocR and we went on to determine the crystal structure of the RocC FinO domain in complex with a RocR terminator. This structure provides the first view of any FinO chaperone bound to its RNA and provides the basis for our key hypotheses that will guide our experiments in the next term of this program. Hypothesis: We propose that FinO chaperones bind transcriptional terminator structures in a manner that reads the length of the 3' ssRNA tail. In addition, we propose that the mechanism of binding allows the remodeling of the sRNA to enable sRNA-mRNA pairing. Specific Aim 1. Refining our understanding of RocC-RocR interactions. To understand the functional implications of our RocC-RocR structure, we are constructing a large set of site-directed point mutations in RocC as well as modifications in the RocR hairpin-tail to understand critical interactions for binding affinity/specificity. The effects of these mutations will be probed through biochemical and biophysical assays, and their effects on RocR stabilization, gene expression and competence will be assessed in Legionella. Specific Aim 2. Probing the effects of RocC binding on RocR structure and interactions with mRNA targets. In preliminary work, we showed that RocR adopts a pseudoknot structure between the terminator hairpin and the sRNA "seed" sequence, and we propose that this must unfold to facilitate sRNA-mRNA pairing. We will test this idea through footprinting, NMR studies, crystallization of the pseudoknot structure and analysis of larger scale RocC-RocR structures via small angle X-ray scattering. These hypotheses will be further tested through in vivo studies in Legionella. Specific Aim 3. Understanding the breadth of FinO chaperone function in bacteria. Using a set of FinO chaperones that span the FinO phylogenetic tree, we will compare their structures, RNA binding specificities, and RNA pairing activities to understand the degree of conservation of FinO functions, and these roles will be further tested in domain-swap experiments. These experiments should reveal the basis for specificity differences between these chaperones, as well as how these proteins facilitate RNA-RNA pairing.