STRUCTURE/FUNCTION OF EUKARYOTIC RNASE III

真核 RNA 酶 III 的结构/功能

• 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
• 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

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RNT 1（核糖核酸酶3）基因编码一个双链 来自酵母酿酒酵母的链特异性核糖核酸内切酶。 RNT 1蛋白在结构上与细菌RNase III相似， 功能，并且是酵母中前核糖体RNA加工事件所必需的。 真核生物的pre-rRNA加工也依赖于小的核仁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 这是一个不同寻常的机会来探索这种基本酶在 真核基因表达和RNA生物合成，可能影响mRNA 稳定性或RNA病毒的复制。 一个总体假设， 酵母RNase III是合成所需的， 细胞生长所需的几种不稳定非核糖体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 使用反向遗传学的酵母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）开发研究系统 snoRNA与RNase III协同作用的机制， 前rRNA加工和核糖体组装。 第一个具体目标是 通过研究RNT 1蛋白及其突变体的功能， 确定蛋白质的哪些部分是底物结合所需的， 切割、金属结合和多聚化。 第二个具体目标 基因筛选抑制子和增强子（合成致死突变） 对于RNT 1基因中的两个可用的温度敏感突变， 用于鉴定关键底物，其被RNase III切割对于 细胞生长 第三个具体目标将需要努力复制 在A0中观察到RNase III和U3 snRNA的体内需求 体外处理事件。 总之，拟议的实验应 提供了一个保守的和基本的初始特征， 真核基因产物，对此知之甚少。