POLYADENYLATION AND RNA TURNOVER

该项目的长期目标是了解聚腺苷化过程如何帮助调节原核生物中的RNA衰变。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，聚腺苷酸化发生在细菌中，是降解mRNA和rRNA的转录后机制的一个组成部分。我们假设聚腺苷化通过靶向某些mRNA和rRNA物种的衰变来帮助细胞快速适应环境的变化。例如，以这种方式，细胞可以经历从对数生长到固定阶段的平滑过渡。为了验证这一假设，我们计划：1。确定当细胞必须适应不同的生长条件时，聚腺苷化活性是如何变化的；2. 确定改变细胞内聚(a)水平的生理后果；3. 确定在缺乏聚(A)聚合酶I的情况下聚腺苷酸化的机制；4. 使用标记的poly(A)聚合酶I、Rnase II和多核苷酸磷酸化酶纯化和表征与mrna 3'端结合的潜在多蛋白复合物；5. 使用我们的体外测定系统确定poly（）依赖性mRNA衰变的生化成分；和6。确定是否存在聚腺苷化靶序列。通过本应用程序中描述的实验，我们希望在分子水平上对原核生物中聚腺苷酸化的功能有更深入的了解。如果细菌细胞需要正常的聚腺苷酸化来适应不断变化的生长环境，那么该系统的成分可能被证明是新的抗菌剂的有吸引力的靶标。

The long range goal of this project is to understand how the process of polyadenylation helps regulate RNA decay in prokaryotes. Long considered a feature unique to eukaryotic organisms, recent work has demonstrated unequivocally that polyadenylation occurs in bacteria and is an integral part of the post-transcriptional machinery that degrades both mRNA and rRNA. We hypothesize that polyadenylation helps cells rapidly adapt to changes in their environment by targeting certain mRNA and rRNA species for decay. In this fashion, for example, a cell can undergo a smooth transition from logarithmic growth to stationary phase. In order to test this hypothesis, we plan to: 1. Determine how polyadenylation activity varies when cells have to adapt to different growth conditions; 2. Determine the physiological consequences of altering poly(a) levels within the cell; 3. Determine the mechanism of polyadenylation in the absence of poly(A)polymerase I; 4. Purify and characterize potential multi-protein complexes that bind to 3' termini of mRNAs using tagged poly(A) polymerase I, Rnase II and polynucleotide phosphorylase; 5. Determine the biochemical components of poly() dependent mRNA decay using our in vitro assay system; and 6. Determine if there are polyadenylation target sequences. With the experiments described in this application, we hope to obtain a more sophisticated understanding at the molecular level of the function of polyadenylation in prokaryotes. If normal polyadenylation is required for bacterial cells to adapt to changing growth environments, components of the system may prove to be attractive targets for new antimicrobials.