Characterization of mRNA Instability Determinants

基因表达的整个过程涉及几个事件 包括转录，RNA加工，mRNA降解， 翻译和翻译后蛋白质修饰。 非常 关于mRNA的机制或调节知之甚少， 降解 拟议实验的重点是 表征mRNA不稳定性序列，其决定快速的 不稳定mRNA的衰变率。 一个不稳定决定因素 从大肠大肠杆菌氯霉素乙酰转移酶基因已被 鉴定的（287 R），可以顺式显性方式发挥作用， 使通常稳定的ompA mRNA不稳定。 几种方法将 用于表征287 R不稳定性决定因素。 第一章 将在序列中引入缺失以鉴定 最小的序列仍然具有破坏稳定的活性。 2)研究中心- 定向诱变将用于进一步确定最小 不稳定序列，并探讨功能的重要性， 序列中的潜在结构。 3)核糖核酸酶， 将识别这种不稳定序列。 最初 将在突变体中测试不稳定序列的功能 在已知的核糖核酸酶中有缺陷。 如果这些实验表明 一种新的核糖核酸酶与287 R序列相互作用，它将 被隔离和特征化。 将进行体外衰变试验， 开发并用于监测酶纯化。 4)如果时间 允许，这种不稳定性决定因素对增长率的影响 对ompA mRNA降解的调控。大肠杆菌亦会进行研究。 %%% 本项目的目标是阐明 细胞降解信使RNA分子。 mRNA降解是一种基本的 代谢过程，对生物体的生存至关重要， 适应能力。 特定mRNA的衰减率可以 影响从转录物合成蛋白质的量 从而对细胞的表达水平产生重要影响。 相应的基因。 本提案中概述的实验将 描述mRNA中的不稳定性决定簇 导致不稳定的mRNA快速衰减。 的序列或 不稳定性功能所需的结构 决定因素将被定义，和细胞酶， 将识别这些序列。

The overall process of gene expression involves several events including transcription, RNA processing, mRNA degradation, translation, and post-translational protein modification. Very little is known concerning the mechanism or regulation of mRNA degradation. The focus of the proposed experiments is to characterize mRNA instability sequences that determine the rapid decay rate of labile mRNAs. An instability determinant originating from the E. coli chloramphenicol acetyltransferase gene has been identified (287R) that can function in a cis-dominant manner to destabilize the normally stablw ompA mRNA. Several approaches will be used to characterize the 287R instability determinant. 1) Deletions will be introduced into the sequence to identify the smallest sequence that still has destabilizing activity. 2) Site- directed mutagenesis will be used to further define the minimum instability sequence and to probe the functional importance of potential structures within the sequence. 3) The ribonuclease that recognizes this instability sequence will be identified. Initially the function of the instability sequence will be tested in mutants defective in known ribonucleases. If these experiments indicate that a novel ribonuclease interacts with the 287R sequence, it will be isolated and characterized. An in vitro decay assay will be developed and used to monitor enzyme purification. 4) If time permits, the effect of this instability determinant on growth rate regulation of ompA mRNA decay in E. coli will also be investigated. %%% The goal of this project is to elucidated the mechanisms by which cells degrade messenger RNA molecules. mRNA degradation is a basic metabolic process and is vital to the organism's survival and adaptation abilities. The decay rate of a particular mRNA can influence the amount of protein synthesized from that transcript and thereby have a critical impact on the expression levels of the corresponding gene. The experiments outlined in this proposal will characterize elements within mRNAs called instability determinants that result in the rapid decay of labile mRNAs. The sequences or structures that are required for the function of an instability determinant will be defined, and the cellular enzymes that recognizes these sequences will be identified.