Molecular Basis for mRNA Decay in Bacteria

总结 生物学的一个中心问题是了解细胞如何优化生物过程，以应对变化 环境条件mRNA的衰变机制通过限制细胞的增殖来协调活动。 每个mRNA可以翻译成蛋白质分子的次数。最近的研究发现了一种新的 细菌中由三磷酸化的初始转化触发的5 '末端依赖性mRNA衰变途径 初级转录物的5'末端通过Nuvitamin水解酶RppH转化为单磷酸。5 '端的三磷酸 细菌mRNA的帽状结构具有类似于真核生物mRNA的帽状结构的保护作用; 因此，通过RppH去除焦磷酸盐与真核生物去帽酶的作用平行。尽管 至关重要的是，5 '-末端依赖性mRNA衰变是基因表达的最不了解的机制之一。 调控该提案详细说明了一系列具体目标，这些目标涉及特异性、催化机制和 RppH和RppH相关因子在调节细菌基因表达中的作用。该提案整合了 X射线晶体学、遗传学、生物化学方法、聚焦全基因组RNA测序和 生物信息学具体目标1致力于确定RppH的催化机理， 了解RppH的底物特异性。本目标将阐述RNA“去帽”的分子原理 基于大肠杆菌RppH结合到模型RNA的晶体结构， 诱变Specific Aim 2将验证5 '-末端依赖性mRNA衰减的新模型。这一目标将 识别和表征参与该途径的新因子及其对基因表达景观的影响。 特异性目标3将在各种条件下在全基因组范围内鉴定RppH的细胞mRNA靶标。 环境条件这一目的将揭示RNA的分子特征，是重要的RppH 识别并揭示RppH对细菌中基因表达控制的贡献。的结果 这些研究将提供一个全面的图片的关键步骤，在5 '端依赖途径， 细菌mRNA的衰减，从而解决了我们对调控网络的理解中的一个基本空白 由RNA降解控制。

Summary A central problem in biology is to understand how cells optimize biological processes in response to changing environmental conditions. mRNA decay mechanisms contribute to coordination of activities by limiting the number of times each mRNA can be translated into protein molecules. Recent studies have identified a novel 5'-end-dependent mRNA decay pathway in bacteria triggered by the initial conversion of the triphosphorylated 5' end of primary transcripts to a monophosphate by the Nudix hydrolase RppH. A triphosphate on the 5'-end of bacterial mRNA has a protective effect analogous to that of the cap structure of eukaryotic mRNAs; therefore, pyrophosphate removal by RppH parallels the action of eukaryotic decapping enzymes. Despite its vital importance, 5'-end-dependent mRNA decay is among the least understood mechanisms of gene regulation. This proposal details a set of specific aims that address specificity, catalytic mechanism, and the role of RppH and RppH-associated factors in regulating gene expression in bacteria. The proposal integrates X-ray crystallography, genetics, biochemical methods, focused genome-wide RNA sequencing and bioinformatics. Specific Aim 1 is devoted to the determination of the catalytic mechanism of RppH and understanding the substrate specificity of RppH. This Aim will address molecular principles of RNA `decapping' based on the crystal structures of Escherichia coli RppH bound to model RNAs and structure-guided mutagenesis. Specific Aim 2 will validate a new model for the 5'-end-dependent mRNA decay. This Aim will identify and characterize new factors involved in the pathway and their impact on gene expression landscape. Specific Aim 3 will identify cellular mRNA targets of RppH on a genome-wide scale under various environmental conditions. This Aim will reveal the molecular features of RNAs that are important for RppH recognition and uncover the contribution of RppH to the control of gene expression in bacteria. The results of these studies will provide a comprehensive picture of the critical steps in the 5'-end-dependent pathway for bacterial mRNA decay and thereby address a fundamental gap in our understanding of the regulatory networks controlled by RNA degradation.