Structural studies of exoribonucleases

描述(申请人提供)：核糖核酸酶(RNase)在所有活细胞中的许多重要的RNA细胞过程中发挥核心作用。其中一个过程是信使核糖核酸的降解，这是转录后调控基因表达的一个重要机制。结构RNA的成熟和周转也需要RNA酶。大肠杆菌已经成为了解核糖核酸酶在细胞RNA代谢中作用的模式系统，已经在这种细菌中鉴定出八种不同的外切核糖核酸酶。其中，三个(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，该超家族包括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家族外切核糖核酸酶。然而，这些外切核糖核酸酶在功能上是完全不同的。我们与迈阿密大学Murray Deutscher博士的实验室合作，开始了对这个外切核糖核酸酶家族的结构研究，以确定这些蛋白质的结构特征。寡核糖核酸酶的结构已经解决，我们得到了RNaseT的衍射性晶体。具体来说，我们建议： 1.对寡核苷酸酶进行详细的结构和功能研究，以更好地了解其活性部位、金属需求和二聚体状态。我们还建议通过获得人类寡核酸酶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.对RNaseT的晶体进行优化，推导出其原子结构。 3.确定核糖核酸酶D的原子结构。 4.了解这三种酶在底物专一性、四级结构、金属需要量和催化机理等方面的异同，以便更好地表征这一家族酶。 这项研究的长期目标是了解单个有机体(大肠杆菌)中所有外切核糖核酸酶的结构和作用机制；这些研究将补充一项平行研究，以完全确定和表征大肠杆菌中所有外切核酸酶的生理作用，目前正在Deutscher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.