The Role of Polyadenylation in RNA Turnover

该项目的长期目标仍然是了解多聚腺苷酸化在转录后修饰中的作用。 原核生物中的调节。虽然长期以来被认为是真核生物独有的特征， 在过去的10年里，已经表明多聚腺苷酸化与mRNA的衰变密切相关，rRNA 在大肠杆菌中的加工、tRNA成熟和总体RNA质量控制。同样重要的是， 现在已经表明，通过聚（A）聚合酶类型的酶或多核苷酸的聚腺苷酸化， 磷酸化酶参与几乎所有原核生物的转录后修饰。此外，本发明还提供了一种方法， 在过去的几年中，已经显示真核生物含有核多腺苷酸化系统， 与E相似。大肠杆菌系统，因为它靶向有缺陷的Pol II转录物进行降解。因此E.杆菌 这是研究这一复杂系统的极好的原核模型系统。根据我们的进展， 在目前的赠款期间，我们现在能够开发一个更复杂的 了解多聚腺苷酸化的生化机制及其在mRNA衰变中的作用， tRNA成熟和rRNA加工。具体的实验方法包括：1.确定是否 PAP I优先聚腺苷酸化全长或部分降解的转录物; 2.确定如何 聚腺苷酸化有助于启动mRNA衰变; 3.生理条件下多聚腺苷酸化复合物的分析 PAP I水平; 4.体外聚腺苷酸化复合物的表征; 5.识别和 固定相特异性聚（A）聚合酶的表征; 6.确定基本特征 使Rho非依赖性转录终止子成为多聚腺苷酸化信号; 7.分析转录后 多顺反子转录物的修饰终止于或不终止于Rho非依赖性 转录终止子; 8.确定多聚腺苷酸化在rRNA和tRNA成熟中的作用; 9.分析聚（A）尾和基于RNase E的降解体在起始聚合酶中的相互作用。 mRNA衰变。由于多聚腺苷酸化似乎存在于广泛的原核生物中，包括许多 病原体，更全面地了解这一系统提供了一个机会，以确定 抗微生物药物的新靶点，其将对革兰氏阴性或革兰氏阳性细菌具有活性。

The long range goal of this project still remains to understand the role that polyadenylation plays in posttranscriptional regulation in prokaryotes. Although long considered to be a feature unique to eukaryotes, over the past 10 years it has been shown that polyadenylation is intimately involved in mRNA decay, rRNA processing, tRNA maturation and overall RNA quality contol in Escherichia coli. Of equal importance, it has now been shown that polyadenylation, either by poly(A) polymerase type enzymes or polynucleotide phosphorylase are involved in the post-transcriptional modification in almost all prokaryotes. In addition, over the past few years it has been shown that eukaryotes contain a nuclear polyadenylation system that closely mimics the E. coli system in that it targets defective Pol II transcripts for degradation. Thus E. coli is an excellent prokaryotic model system for studying this complex system. Based on the progress that we have made during the current grant period, we are now in a position to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles it plays in mRNA decay, tRNA maturation and rRNA processing. Specific experimental approaches include: 1. Determine whether PAP I preferentially polyadenylates full-length or partially degraded transcripts; 2. Determine how polyadenylation helps initiate mRNA decay; 3. Analysis of the polyadenylation complex at physiological levels of PAP I; 4. Characterization of the polyadenylation complex in vitro; 5. Identification and characterization of the stationary phase-specific poly(A) polymerase; 6. Determine the essential features that make a Rho-independent transcription terminator a polyadenylation signal; 7. Analyze the posttranscriptional modification of polycistronic transcripts terminating with or without a Rho-independent transcription terminator; 8. Determine the role of polyadenylation in the maturation of rRNAs and tRNAs; and 9. Analyze the interactions between poly(A) tails and the RNase E based degradosome in the initiation of mRNA decay. Since polyadenylation appears to be present in a wide range of prokaryotes, including many pathogenic organisms, a more complete understanding of this system provides an opportunity to identify new targets for anti-microbials that will be active against either gram negative or gram positive bacteria.