Structural studies of exoribonucleases

描述（由申请人提供）：核糖核酸酶（RNases）在所有活细胞的许多重要RNA细胞过程中起核心作用。其中一个过程是mRNA降解，这是基因表达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。大肠杆菌是了解核糖核酸酶在细胞RNA代谢中的作用的模型系统，在这种细菌中已经鉴定出八种不同的外核糖核酸酶。其中，三个（核糖核酸酶T，核糖核酸酶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，包括DNA聚合酶的校对结构域。这三种蛋白具有相似的序列基序，被称为DEDD家族外核糖核酸酶。然而，这些外核糖核酸酶在功能上是完全不同的。我们与迈阿密大学的Murray Deutscher博士的实验室合作，开始了对这个外核糖核酸酶家族的结构研究，以表征这些蛋白质的结构特征。低核糖核酸酶的结构已经被解开，我们得到了RNase t的衍射质量晶体。具体来说，我们提出：

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.