The Role of tmRNA in development of C. crescentus

描述（由申请人提供）： 缺乏反式翻译活性的细菌在发育、分化、毒力、应激反应和生存力方面存在缺陷，但这些过程需要反式翻译的原因尚不清楚。我们的长期目标是了解细菌中反式翻译的作用机制和生理作用。本申请的总体目标是了解如何使用反式翻译活性和底物选择性的调节来控制新月柄杆菌中负责分化和细胞周期进程的遗传途径。我们的中心假设是细菌调节反式翻译的关键底物的产生，以及tmRNA和SmpB的可用性，以控制负责启动DNA复制和其他生理过程的遗传电路。本研究的目的是建立一个通过控制反式翻译进行转录后调控的范例。本论文的主要目的是：1）明确C.新月形; 2）鉴定调节反式翻译活性的机制;和3）确定反式翻译在共翻译分泌中的作用。根据第一个目标，遗传和生物化学的方法将被用来确定顺式和反式作用因子负责底物的选择性，通过在66%的反式翻译底物中发现的核酸基序。在第二个目标中，将使用遗传和生物化学测定来鉴定负责细胞周期调节的SmpB蛋白水解和RNA酶R降解tmRNA的分子相互作用。在第三个目标中，将测试反式翻译和SecYEG易位蛋白之间遗传相互作用的分子基础。这项研究具有重要意义，因为它将为细菌生长和发育所需的转录后基因调控提供一个范例。这一范例将为更详细地了解细菌如何响应环境和发育线索而快速改变其基因表达谱打开大门。在细菌发育的模型系统中阐明这一过程有望为解释来自其他物种的大量数据提供一个框架，并为理解细菌在环境中和发病过程中如何使用反式翻译提供基础。

DESCRIPTION (provided by applicant): Bacteria lacking trans-translation activity have defects in development, differentiation, virulence, stress responses, and viability, but the reasons trans-translation is required for these processes are not known. Our long-term goal is to understand the mechanism of action and physiological role of trans-translation in bacteria. The overall objective of this application is to understand how regulation of trans-translation activity and substrate selectivity is used to control genetic pathways responsible for differentiation and cell cycle progression in Caulobacter crescentus. Our central hypothesis is that bacteria regulate the generation of key substrates for trans-translation, as well as the availability of tmRNA and SmpB, to control genetic circuits responsible for initiation of DNA replication and other physiological processes. The rationale for the proposed research is to establish a paradigm for post-transcriptional regulation through controlled trans-translation. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the mechanism for generation of trans-translation substrates in C. crescentus; 2) identify mechanisms for regulation of trans- translation activity; and 3) determine the role of trans-translation in co-translational secretion. Under the first aim, genetic and biochemical approaches will be used to identify cis- and trans-acting factors responsible for substrate selectivity through a nucleic acid motif found in 66% of trans-translation substrates. In the second aim, genetic and biochemical assays will be used to identify the molecular interactions responsible for cell- cycle regulated SmpB proteolysis and tmRNA degradation by RNase R. In the third aim, the molecular basis for the genetic interaction between trans-translation and the SecYEG translocator will be tested. The proposed research is significant because it will provide a paradigm for post-transcriptional gene regulation by trans- translation that is required for bacterial growth and development. This paradigm will open the door to a more detailed understanding of how bacteria rapidly change their gene expression profiles in response to environmental and developmental cues. Elucidation of this process in a model system for bacterial development is expected to provide a framework for interpreting a wide array of data from other species, and a basis for understanding how trans-translation is used by bacteria in the environment and during pathogenesis.