Effectors and regulators of end-dependent mRNA degradation in bacteria

描述（由申请人提供）：所有细菌都依赖mRNA降解作为调节基因表达和适应不断变化的生存环境的手段。对于许多转录本来说，快速降解是由5'端的磷酸化状态控制的，如果单磷酸化，它可以使RNA的衰变增强近一个数量级。RppH最近被确定为触发5‘端依赖性降解的酶，通过从初级转录本的5’端去除磷酸盐，产生短寿命的5'单磷酸化衰变中间体；然而，我们对RppH在细胞RNA代谢的大背景下的作用的认识仍然很初级。本研究的主要目的是利用大肠杆菌和枯草芽孢杆菌作为模式生物，进一步了解细菌中5'端依赖性RNA衰变。为了实现这一目标，将创建一个报告系统来检测5'端依赖性mRNA降解的变化，并使用正向遗传学来：1。确定焦磷酸水解酶活性的其他来源；和2。发现RppH的正、负调节因子。有希望的候选者将通过生化和分子生物学分析进一步表征，以确定它们对5'端依赖性RNA衰变的影响范围和机制。这些研究的结果将增强我们对细菌如何利用RNA降解来控制其基因表达谱的理解，并为已知影响细菌发病机制的调节过程提供见解。

DESCRIPTION (provided by applicant): All bacteria rely on mRNA degradation as a means to regulate gene expression and to adapt to the ever changing milieu in which they live. For a number of transcripts, rapid degradation is governed by the phosphorylation state of the 5' end, which can potentiate RNA decay by nearly an order of magnitude if monophosphorylated. RppH was recently identified as the enzyme responsible for triggering 5' end-dependent degradation by removing phosphates from the 5' end of primary transcripts to produce short-lived 5' monophosphorylated decay intermediates; however, our knowledge of the role of RppH in the larger context of cellular RNA metabolism remains quite rudimentary. The primary purpose of this proposal is to further our understanding of 5' end-dependent RNA decay in bacteria, using Escherichia coli and Bacillus subtilis as model organisms. To achieve this goal, a reporter system will be created to detect changes in 5' end- dependent mRNA degradation, and forward genetics will be used to: 1. identify additional sources of pyrophosphohydrolase activity; and 2. discover positive and negative regulators of RppH. Promising candidates will be further characterized by biochemical and molecular biological assays to determine the scope and mechanism of their influence on 5' end-dependent RNA decay. The results of these studies will enhance our understanding of how bacteria use RNA degradation to control their gene expression profile and provide insights into a regulatory process known to influence bacterial pathogenesis.