The Role of tmRNA in development of C. crescentus

描述(由申请人提供)： 缺乏转译活性的细菌在发育、分化、毒力、应激反应和生存能力方面存在缺陷，但这些过程需要转译的原因尚不清楚。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。这一应用的总体目标是了解如何利用反式翻译活性和底物选择性的调节来控制新月弯杆菌的分化和细胞周期进展的遗传途径。我们的中心假设是细菌调节转译关键底物的产生，以及tmRNA和SmpB的可获得性，以控制负责启动DNA复制和其他生理过程的遗传电路。这项研究的理论基础是通过受控翻译为转录后调控建立一个范例。中心假设将通过追求以下具体目标来验证：1)确定新月浑藻反译底物的产生机制；2)确定反译活动的调节机制；以及3)确定反译在共翻译分泌中的作用。在第一个目标下，将使用遗传和生化方法，通过在66%的反式翻译底物中发现的核酸基序来识别负责底物选择性的顺式和反式作用因子。在第二个目标中，将利用遗传和生化分析来确定RNaseR对细胞周期调节的SmpB蛋白降解和tmRNA降解的分子相互作用。第三个目标，将测试反式翻译和SecYEG转运子之间遗传相互作用的分子基础。这项拟议的研究具有重要意义，因为它将为细菌生长和发育所需的转录后基因转录后调控提供一个范例。这一范式将为更详细地了解细菌如何根据环境和发育线索迅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提供一个框架，用于解释来自其他物种的广泛数据，并为理解细菌在环境中和致病过程中如何使用反式翻译奠定基础。

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.