The Role of Polyadenylation in RNA Turnover

描述（由申请人提供）：该项目的长期目标是了解聚腺苷化在原核生物转录后调控中的作用。长期以来被认为是真核生物独有的特征，最近的研究明确表明，聚腺苷化在mRNA衰变、rRNA加工和rRNA降解中都起着不可或缺的作用。我们已经假设，聚腺苷化通过靶向某些mRNA和rRNA物种的衰变，帮助细胞快速适应环境的变化。由于在大量的原核生物中发现了聚(A)聚合酶的同源物，大肠杆菌是研究这一复杂系统的一个很好的模型系统。本文描述的实验旨在帮助阐明聚腺苷酸化的分子机制，并对其在细胞生长和存活中的功能有更深入的了解。具体的思路包括：1。分析聚(A)聚合酶I、多核苷酸磷酸化酶和Hfq蛋白与其mRNA底物的相互作用；2. 确定mRNA聚腺苷化信号的基本特征；3. 固定相特异性聚(A)聚合酶的克隆与鉴定4. 确定多核苷酸磷酸化酶在聚腺苷化和mRNA衰变途径中的功能；5. 确定多磷酸激酶（Ppk）是否在调节聚腺苷酸化途径中起作用；6. 确定聚腺苷化是否影响细胞适应不同生长条件；7. 鉴定和表征潜在的多聚(A)结合蛋白的作用，包括CspE；和8。确定聚腺苷化是否影响翻译效率。由于与真核生物相比，聚腺苷酸化在大肠杆菌中的作用似乎非常不同，因此更好地了解该系统的工作原理非常重要。这项工作的另一个潜在好处是可能确定抗菌素的新靶点。

DESCRIPTION (provided by applicant): The long-range goal of this project is to understand the role that polyadenylation plays in prokaryotic post-transcriptional regulation. Long considered a feature unique to eukaryotic organisms, recent work has demonstrated unequivocally that polyadenylation plays an integral role in both mRNA decay, rRNA processing and rRNA degradation. We have hypothesized that polyadenylation helps cells rapidly adapt to changes in their environment by targeting certain mRNA and rRNA species for decay. Since poly(A) polymerase homologues are found in a large number of prokaryotes, Escherichia coli is an excellent model system for studying this complex system. The experiments described here are designed to help elucidate the molecular mechanism of polyadenylation and to derive a more sophisticated understanding of its function in the growth and survival of the cell. Specific lines of approach include: 1. Analyze the interactions of poly(A) polymerase I, polynucleotide phosphorylase and the Hfq protein with its mRNA substrates; 2. Determine the essential features of mRNA polyadenylation signals; 3. Clone and characterize the stationary phase-specific poly(A) polymerase; 4. Determine the function of polynucleotide phosphorylase in the polyadenylation and mRNA decay pathways; 5. Determine if polyphosphate kinase (Ppk) plays a role in regulating the polyadenylation pathway; 6. Determine whether polyadenylation affects how cells adapt to different growth conditions; 7. Identify and characterize the role of potential poly(A) binding proteins, including CspE; and 8. Determine if polyadenylation affects translation efficiency. Since polyadenylation seems to function very differently in E. coli compared to eukaryotic organisms, it is important to develop a better understanding of how this system works. An additional potential benefit from this work is the possible identification of new targets for antimicrobials.