Investigating the mechanism of mRNA cleavage in the ribosomal A-site

项目描述（申请人提供）：该项目的具体目标是阐明RelE诱导a位点mRNA裂解的催化机制。在应激条件下，RelE诱导a位点mRNA的密码子特异性切割，抑制真细菌和古细菌的翻译。a位点mRNA的切割并不是RelE所独有的。这种分裂在核糖体停滞时被观察到，并且被认为是核糖体对停滞核糖体的再循环所必需的；然而，解理所需的因素是未知的。RelE对a位点mRNA的密码子特异性识别与释放因子观察到的相似。该项目的总体目标是确定a位点mRNA的切割是如何发生的，以深入了解这种形式的翻译调节。本提案的具体目标是确定a位点裂解的化学性质（目的1），研究催化金属离子的存在（目的2），并探索残基在RelE和核糖体中的催化作用（目的3和4）。由于RelE不能切割裸露的mRNA，不含预期的催化酸或碱，并且核糖体在没有外部因素的情况下不会在a位点切割mRNA，因此尚不清楚核糖体或RelE是否含有mRNA切割所需的催化成分。具有开放a位点的停滞70S核糖体复合物将用于体外监测mRNA的切割。将确定裂解mRNA的特定5‘和3’产物（目的1），确定反应化学并为可能的催化机制提供线索。金属离子特异性开关实验将用于识别催化金属离子的存在。relle中保守残基的丙氨酸突变体，以及a位点残基的非致死核糖体突变体，将使用单次翻转动力学进行分析，以确定催化残基是否位于relle或核糖体上（目的3和4）。结合和催化的信息将阐明核糖体和RelE在mRNA切割中的个体贡献。公共卫生相关性：本建议概述了核糖体循环的第一步，a位点mRNA切割的调查。一些抗生素通过阻止核糖体和阻断翻译和a位点切割而起作用，这与抗生素耐药性有关。了解a位点mRNA切割的机制可能有助于了解如何更有效地使用抗生素来阻断翻译。

DESCRIPTION (provided by applicant): The specific goal of this project is to elucidate the catalytic mechanism of A-site mRNA cleavage induced by RelE. Under stressful conditions, RelE induces codon specific cleavage of mRNA in the A-site, inhibiting translation in eubacteria and archae. A-site mRNA cleavage is not unique to RelE. This cleavage has been observed upon ribosome stalling and is thought to be required for ribosome recycling of stalled ribosomes; however, the factors required for cleavage are unknown. Codon specific recognition of A-site mRNA by RelE is similar to that observed by the release factors. The broad goals of this project are to determine how A-site mRNA cleavage occurs to gain insight into this form of translation regulation. The specific goals of this proposal are to determine the chemical nature of A-site cleavage (aim 1), investigate the presence of catalytic metal-ions (aim 2), and explore the catalytic role of residues in RelE and the ribosome (aims 3 and 4). Because RelE cannot cleave naked mRNA and does not contain the expected catalytic acid or base and the ribosome does not cleave mRNA in the A-site in the absence of external factors, it is unclear if the ribosome or RelE contains the catalytic components necessary for mRNA cleavage. Stalled 70S ribosomes complexes with an open A-site will be used to monitor mRNA cleavage in vitro. The specific 5' and 3' products of the cleaved mRNA will be determined (aim 1), identifying the reaction chemistry and providing clues to possible catalytic mechanism. Metal-ion specificity switch experiments will be used to identify the presence of catalytic metal-ions. Alanine mutants of conserved residues in RelE, as well as non-lethal ribosome mutants of residues in the A-site, will be assayed using single turnover kinetics to identify if catalytic residues reside on RelE or the ribosome (aim 3 and 4). Information on binding and catalysis will illuminate the individual contributions of the ribosome and RelE in cleavage of mRNA. PUBLIC HEALTH RELEVANCE: This proposal outlines the investigation of initial step in ribosomal recycling, A-site mRNA cleavage. Several antibiotics work by stalling the ribosome and blocking translation and A-site cleavage has been linked to antibiotic resistance. Understanding the mechanism of A-site mRNA cleavage may add insight into how to more efficiently use antibiotics to block translation.