Structural studies of exoribonucleases

描述（由申请人提供）：核糖核酸酶（RNase）在所有活细胞中的许多重要RNA细胞过程中发挥核心作用。这些过程之一是mRNA降解，这是基因表达的转录后控制的重要机制。RNA酶也是结构RNA成熟和周转所必需的。E.大肠杆菌作为一个模型系统，了解核糖核酸酶的作用，在细胞RNA代谢，和八个不同的外切核糖核酸酶已被确定在这种细菌。其中，三种（RNase T，RNase D和寡核糖核酸酶）是一个更大的核酸外切酶超家族的成员，该家族包括DNA聚合酶的校对结构域。这三种蛋白质具有相似的序列基序，被称为DEDD家族核糖核酸外切酶。然而，这些核糖核酸外切酶在功能上是相当不同的。我们与迈阿密大学的Murray Deutscher博士的实验室合作，开始了对这个核糖核酸外切酶家族的结构研究，以从结构上表征这些蛋白质。寡核糖核酸酶的结构已经解决，我们有RNase T的衍射质量晶体。具体而言，我们建议： 1.启动寡核糖核酸酶的详细结构-功能研究，以更好地了解其活性位点、金属需求和二聚体状态。我们还建议通过获得寡核糖核酸酶的人类同源物的结构来寻找这种酶的原核和真核形式之间的差异。 2.优化RNase T获得的晶体，并推导出其原子结构。 3.确定RNase D的原子结构。 4.了解这三种酶在底物特异性、四级结构、金属需求和催化机制方面的异同，更好地表征这一酶家族。 这项研究的长期目标是了解单个生物体中所有核糖核酸外切酶的结构和作用机制（E。coli）;这些研究将补充一个平行的研究，以完全确定和表征所有的外切核糖核酸酶在大肠杆菌中的生理作用。大肠杆菌，目前正在德国实验室进行。

DESCRIPTION (provided by applicant): Ribonucleases (RNases) play a central role in a number of vital RNA cellular processes in all living cells. One of these processes is mRNA degradation, which is an important mechanism for post-transcriptional control of gene expression. RNases are also required for maturation and turnover of structural RNAs. E. coli has served as a model system for understanding the role of ribonucleases in cellular RNA metabolism, and eight distinct exoribonucleases have been identified in this bacterium. Of these, three (RNase T, Rnase D, and oligoribonuclease) are members of a larger exonuclease superfamily that includes the proof-reading domains of DNA polymerases. These three proteins share similar sequence motifs and have been dubbed the DEDD family exoribonucleases. However, functionally these exoribonuclease are quite distinct. We have initiated structural studies of this family of exoribonucleases, in collaboration with the laboratory of Dr. Murray Deutscher at the University of Miami, to structurally characterize these proteins. The structure of oligoribonuclease has been solved and we have diffraction quality crystals of RNase T. Specifically, we propose to: 1. Initiate detailed structure-function studies of oligoribonuclease to better understand its active site, metal requirements, and dimeric state. We also propose to look for differences between the prokaryotic and eukaryotic forms of this enzyme, by obtaining the structure of the human homologue of oligoribonuclease. 2. Optimize the crystals obtained for RNase T, and derive its atomic structure. 3. Determine the atomic structure of RNase D. 4. Understand the similarities and differences between these three enzymes in terms of substrate specificity, quarternary structure, metal requirements and catalytic mechanism, to better characterize this family of enzymes. The long term goals of this research are to understand the structures and mechanisms of action of all the exoribonucleases in a single organism (E. coli); these studies will complement a parallel study to completely determine and characterize the physiological role of all the exoribonucleases in E. coli, now underway in the Deutscher laboratory.