STRUCTURE/FUNCTION OF EUKARYOTIC RNASE III

真核 RNA 酶 III 的结构/功能

基本信息

批准号：
2910298
负责人：
Manuel Ares
金额：
$ 13.47万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2001-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2910298
关键词：
cell growth regulation enzyme mechanism enzyme structure enzyme substrate gene expression genetic enhancer element molecular biology protein structure function ribonuclease III ribosomal RNA ribosomes small nuclear RNA suppressor mutations yeasts

项目摘要

DESCRIPTION: The essential RNT1 (ribonuclease three) gene encodes a double strand-specific endoribonuclease from the yeast Saccharomyces cerevisiae. The RNT1 protein appears similar to bacterial RNase III in structure and function and is required for pre-ribosomal RNA processing events in yeast. Eukaryotic pre-rRNA processing is also dependent on small nucleolar RNAs, and at least one processing event is dependent on both yeast RNase III and U3 snoRNA. In bacteria, RNase III is involved in a wide variety of RNA processing events; thus the identification of the enzyme in yeast offers an unusual opportunity to explore the role of this fundamental enzyme in eukaryotic gene expression and RNA biogenesis, where it may influence mRNA stability or the replication of RNA viruses. An overarching hypothesis to be tested is that yeast RNase III is required for the synthesis or degradation of several unstable nonribosomal RNAs necessary for cell growth. In addition, the mechanism of snoRNA facilitation of RNase III activity deserves investigation. Three aims are proposed: 1) To characterize the structure, function and substrate specificity of the yeast RNase III enzyme using reverse genetics and biochemistry; 2) to identify and characterize substrates of RNase III whose processing is key to cell growth; and 3) To develop systems to study the mechanism by which snoRNAs function in concert with RNase III in pre-rRNA processing and ribosome assembly. The first specific aim will be addressed by studying the function of RNT1 protein and its mutants to determine which parts of the protein are required for substrate binding, cleavage, metal binding and multimerization. In the second specific aim genetic screens for suppressors and enhancers (synthetic lethal mutations) for two available temperature sensitive mutations in the RNT1 gene will be used to identify key substrates whose cleavage by RNase III is essential for cell growth. The third specific aim will require an effort to reproduce the observed in vivo requirement for both RNase III and the U3 snRNA in the A0 processing event in vitro. Together, the proposed experiments should provide an initial characterization of a conserved and fundamental eukaryotic gene product about which little is known.

描述：必需的 RNT1（核糖核酸酶三）基因编码一个双来自酿酒酵母的链特异性核糖核酸内切酶。 RNT1 蛋白在结构上与细菌 RNase III 相似，并且功能，并且是酵母中核糖体前 RNA 加工事件所必需的。真核前 rRNA 加工也依赖于小核仁 RNA，并且至少一个加工事件依赖于酵母 RNase III 和 U3 snoRNA。在细菌中，RNase III 参与多种 RNA 处理事件；因此，酵母中酶的鉴定提供了一种探索这种基本酶的作用的难得机会真核基因表达和 RNA 生物发生，可能影响 mRNA RNA病毒的稳定性或复制。一个总体假设需要测试的是酵母 RNase III 是合成所必需的，或者细胞生长所需的几种不稳定的非核糖体 RNA 的降解。此外，snoRNA促进RNase III活性的机制值得调查。提出了三个目标：1）表征结构、功能和使用反向遗传学研究酵母 RNase III 酶的底物特异性和生物化学； 2) 鉴定和表征 RNase III 的底物其加工过程是细胞生长的关键； 3）开发系统进行研究 snoRNA 与 RNase III 协同作用的机制 rRNA 前体加工和核糖体组装。第一个具体目标是通过研究 RNT1 蛋白及其突变体的功能来解决这一问题确定底物结合需要蛋白质的哪些部分，裂解、金属结合和多聚化。在第二个具体目标中抑制子和增强子的遗传筛选（合成致死突变）对于 RNT1 基因中的两个可用的温度敏感突变，将是用于鉴定关键底物，其被 RNase III 切割对于细胞生长。第三个具体目标需要努力重现观察到 A0 中对 RNase III 和 U3 snRNA 的体内需求体外处理事件。总之，所提出的实验应该提供保守和基本的初步表征人们知之甚少的真核基因产物。