Molecular Mechanisms of mRNA Turnover

特定mRNA的衰减率可以极大地影响由该信息产生的蛋白质的数量，因此可以在基因表达的整体调节中发挥重要作用。该项目的目标是阐明细胞降解信使RNA分子的机制，并能够调节这一衰变过程。拟议的实验将确定大肠杆菌氯霉素乙酰转移酶（cat）信息降解的机制，以及该信息的稳定性如何在培养生长条件下受到调节。在cat信息中检测到三个裂解位点，这可能与启动衰变过程有关。这些裂解位点将被绘制出来，这些裂解对衰变过程的重要性将通过缺失和位点导向突变以及基因融合分析来确定。体外信息降解试验将用于研究衰变过程，并最终纯化参与这些降解事件的核糖核酸酶。还将测量cat消息在响应非稳态生长条件和培养基组成时的稳定性变化，并将其与其他消息（如ompA、bla、lpp、lac和trxA）进行比较。这些实验的结果将定义cat信息衰减途径的重要组成部分，这应该广泛适用于大肠杆菌中的其他信息，并将深入了解如何根据培养生长条件调节信息稳定性。

The decay rate of a particular mRNA can greatly influence the amount of protein produced from that message and can thereby play an important role in the overall regulation of gene expression. The goal of this project is to elucidate the mechanisms by which cells degrade messenger RNA molecules and are able to regulate this decay process. The proposed experiments will determine the mechanism by which the E. coli chloramphenicol acetyltransferase (cat) message is degraded and how the stability of this message is regulated in response to culture growth conditions. Three cleavage sites have been detected within the cat message that may be involved in initiating the decay process. These cleavage sites will be mapped, and the importance of these cleavages to the decay process will be determined by deletion and site-directed mutagenesis and by gene fusion analyses. An in vitro message degradation assay will be used to study the decay process and to eventually purify the ribonucleases involved in these degradative events. Changes in the stability of the cat message in response to non-steady state growth conditions and medium composition will also be measured and compared to other messages such as ompA, bla, lpp, lac, and trxA. The results of these experiments will define the important components of the cat message decay pathway, which should be broadly applicable to other messages in E. coli, and will be lend insight into how message stability is regulated in response to culture growth conditions.