Molecular Basis for mRNA Decay in Bacteria

 描述（由申请人提供）：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不断变化的环境条件。mRNA衰变机制通过限制每个mRNA可以翻译成蛋白质分子的次数来协调活动。最近的研究已经确定了细菌中一种新的5 '末端依赖性mRNA衰变途径，该途径由初级转录物的三磷酸化5'末端通过Nuestrophin水解酶RppH初始转化为单磷酸触发。细菌mRNA 5 '端的三磷酸具有类似于真核mRNA帽结构的保护作用;因此，通过RppH去除焦磷酸与真核去帽酶的作用平行。尽管其至关重要，5 '末端依赖性mRNA衰变是最不了解的基因调控机制之一。该提案详细说明了一系列具体目标，这些目标涉及特异性、催化机制以及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，重点全基因组RNA测序和生物信息学。具体目标1致力于确定RppH的催化机制，并了解RppH的底物特异性。本目标将根据大肠杆菌RppH与模型RNA结合的晶体结构和结构指导的诱变来解决RNA "去帽"的分子原理。Specific Aim 2将验证5'-末端依赖性mRNA衰减的新模型。该目标将确定和表征参与该途径的新因素及其对基因表达景观的影响。特定目标3将在各种环境条件下在全基因组范围内识别RppH的细胞mRNA靶标。该目的将揭示RppH识别的重要RNA的分子特征，并揭示RppH对细菌基因表达控制的贡献。这些研究的结果将提供细菌mRNA衰变的5 '末端依赖性途径中关键步骤的全面描述，从而解决我们对RNA降解控制的调控网络的理解中的根本差距。

 DESCRIPTION (provided by applicant): 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.