Characterization of mRNA Instability Determinants

基因表达的整个过程包括转录、RNA加工、mRNA降解、翻译和翻译后蛋白修饰等几个事件。我们对mRNA降解的机制和调控知之甚少。提出的实验的重点是表征mRNA不稳定序列，确定不稳定mRNA的快速衰减率。来自大肠杆菌氯霉素乙酰转移酶基因（287R）的不稳定性决定因素已被确定，该基因可以顺式显性方式作用，使通常稳定的ompA mRNA不稳定。几种方法将用于表征287R不稳定行列式。1)在序列中引入删除，以识别仍然具有不稳定活性的最小序列。2)位点定向诱变将用于进一步确定最小不稳定序列，并探索序列内潜在结构的功能重要性。3)识别该不稳定性序列的核糖核酸酶将被识别。最初，不稳定序列的功能将在已知核糖核酸酶缺陷的突变体中进行测试。如果这些实验表明一个新的核糖核酸酶与287R序列相互作用，它将被分离和表征。将开发一种体外衰变试验，用于监测酶的纯化。4)如果时间允许，还将研究这种不稳定性决定因素对大肠杆菌中ompA mRNA衰变生长速率调节的影响。这个项目的目标是阐明细胞降解信使RNA分子的机制。mRNA的降解是一个基本的代谢过程，对生物体的生存和适应能力至关重要。特定mRNA的衰减率可以影响从该转录本合成的蛋白质的数量，从而对相应基因的表达水平产生关键影响。本提案中概述的实验将表征mrna中称为不稳定决定因素的元素，这些因素导致不稳定mrna的快速衰减。不稳定性决定因子功能所需的序列或结构将被定义，识别这些序列的细胞酶将被识别。

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.