RNA-Protein Interactions in Bacterial RNase P

这项研究的长期目标是了解大肠杆菌RNase P中RNA-蛋白质相互作用，RNase P是一种将前体tRNA（ptRNAs）加工成成熟形式所必需的酶。 E.大肠杆菌核糖核酸酶P是一种核糖核蛋白复合物，由一个催化RNA亚基（M1 RNA）和一个蛋白质辅因子（C5蛋白）组成，这两个亚基对其体内催化活性都是必不可少的。 C5蛋白在RNase P催化中的作用与其他蛋白质促进的RNA催化反应不同，因为它增强了催化RNA的催化效率和多功能性，该催化RNA反式作用于许多底物。 最近的研究表明，该蛋白不仅增强了底物对催化RNA亚基的亲和力，而且还增加了RNA催化切割反应的速率。 关于这种独特的催化RNP复合物的作用机制的几个问题仍然没有答案。 在这个项目中，生物化学和生物物理方法的组合将被用来确定C5蛋白在组装功能性RNase P复合物中的作用。 本研究的第一个目的是采用基于结构的诱变和遗传互补测定来鉴定C5蛋白中对其体内功能至关重要的氨基酸残基。 第二个目标是剖析蛋白质辅因子对底物识别和催化作用的机制。 最后，通过合理设计在蛋白质分子的不同位置带有半胱氨酸残基的C5突变体，并用巯基特异性交联、足迹法和光谱探针修饰半胱氨酸残基，获得了关于（i）C5蛋白和M1 RNA，和（ii）C5蛋白和ptRNA底物之间接触位点的低分辨率信息，将获得并用于获得对于阐明RNA酶P的作用机制至关重要的结构观点。RNA-蛋白质相互作用在体内许多调节系统中起重要作用（例如，基因表达的翻译控制）。 从这项研究的结果将作为一个范例，了解其他RNP控制各种原核和真核细胞过程中的分子间相互作用。 上述大部分实验正在进行中，将迅速提供有价值的见解。 除了为几名本科生和研究生整合实验室研究和教育外，该项目还帮助更新和培养了几项学术合作。

0091081 Venkat GopalanThe long term goal of this research is to understand RNA-protein interactions in Escherichia coli RNase P, an enzyme essential for the processing precursor tRNAs (ptRNAs) to their mature forms. E. coli RNase P, a ribonucleoprotein (RNP) complex, consists of a catalytic RNA subunit (M1 RNA) and a protein cofactor (C5 protein); both subunits are essential for its catalytic activity in vivo. The role of C5 protein in RNase P catalysis is distinct from other protein-facilitated RNA-catalyzed reactions in that it enhances the catalytic efficiency and versatility of a catalytic RNA which acts in trans on numerous substrates. Recent studies have demonstrated that the protein not only enhances the affinity of the substrate for the catalytic RNA subunit but also increases the rate of the RNA-catalyzed cleavage reaction. Several questions with regard to the mechanism of action of this unique catalytic RNP complex remain unanswered. In this project, a combination of biochemical and biophysical approaches will be used to determine the role of C5 protein in assembling a functional RNase P complex. The first objective of this study will be to employ structure-based mutagenesis and a genetic complementation assay to identify amino acid residues in C5 protein that are essential for its function in vivo. The second objective is to dissect the mechanisms by which the protein cofactor can exert effects on substrate recognition as well as catalysis. Lastly, by rational design of C5 mutants bearing cysteine residues at various positions in the protein molecule and modifying the cysteine residues with thiol-specific crosslinking, footprinting and spectroscopic probes, low resolution information regarding contact sites between (i) C5 protein and M1 RNA, and (ii) C5 protein and ptRNA substrates, will be obtained and used to gain structural perspectives critical for elucidating the mechanism of action of RNase P. RNA-protein interactions play an important role in numerous regulatory systems in vivo (e.g., translational control of gene expression). Results from this study will serve as a paradigm for understanding intermolecular interactions in other RNPs that control various prokaryotic and eukaryotic cellular processes. Most of the experiments described above are in progress and will rapidly furnish valuable insights. In addition to integrating laboratory research and education for several undergraduate and graduate students, this project has already helped renew and nurture several academic collaborations.