TRNA SPLICING ENDONUCLEASE IN YEAST

酵母中的 TRNA 剪接核酸内切酶

• 批准号: 3297726
• 负责人: MICHAEL R CULBERTSON
• 金额: $ 15.18万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297726
• 关键词: RNA biosynthesis; RNA splicing; Saccharomyces; alleles; chemical binding; endonuclease; enzyme mechanism; enzyme structure; fungal genetics; gene complementation; gene expression; gene interaction; gene mutation; genetic manipulation; immunoelectron microscopy; ligase; microscopy; molecular cloning; molecular genetics; nucleic acid structure; radioimmunoassay; structural genes; transfer RNA

Splicing of intervening sequences from precursor-tRNA requires at least two functions, an endonuclease and a ligase. These enzymes catalyze excision of the introns and rejoining of the tRNA half-molecules through recognition of conserved features of tRNA structure. Despite the fact that tRNA splicing is readily amenable to genetic and biochemical analysis, little is known about the regulatory and structural genes involved, the potential interaction of proteins or other components that might form a splicing complex, or the possible relationship between splicing and transport of tRNA from nucleus to cytoplasm. Information on these aspects of tRNA splicing have not been forthcoming due to the intractable nature of the membrane-associated tRNA-splicing endonuclease. Previous attempts in several laboratories to study the endonuclease by either forward or reverse genetic techniques have not been successful. Recently, however, mutations in SEN (splicing endonuclease) genes were identified in this laboratory that cause partial deficiencies of endonuclease activity in the yeast Saccharomyces cerevisiae. One mutant also causes pre- tRNA steady-state accumulation and temperature-sensitive growth. By analyzing SEN genes and their products using genetic, biochemical, and immunological approaches the research in this proposal will provide critical information on the structure, organization, function, and intracellular location of tRNA splicing components. The most immediate question to be answered centers on which products are structural components of the endonuclease. Once this central question is answered it should be possible to address the more complex issues of how and where tRNA splicing occurs in intact cells.

前体 tRNA 的插入序列的剪接需要 至少有两种功能：核酸内切酶和连接酶。 这些 酶催化内含子的切除和内含子的重新连接 通过识别 tRNA 半分子的保守特征 tRNA 结构。 尽管 tRNA 剪接很容易 适合遗传和生化分析，但知之甚少 关于所涉及的调控和结构基因，潜力 蛋白质或其他成分的相互作用可能形成 剪接复合物，或剪接和剪接之间可能的关系 tRNA从细胞核转运至细胞质。 有关信息 tRNA 剪接的这些方面尚未实现，因为 膜相关 tRNA 剪接的棘手本质 核酸内切酶。 此前多个实验室曾尝试进行研究 通过正向或反向遗传技术进行内切核酸酶 还没有成功。 然而最近，SEN 突变 本实验室鉴定了（剪接核酸内切酶）基因 导致核酸内切酶活性部分缺陷 酿酒酵母。 一种突变体也会导致预 tRNA 稳态积累且温度敏感 生长。 通过利用遗传学分析SEN基因及其产物， 生物化学和免疫学方法在这方面的研究 提案将提供有关结构的关键信息， tRNA 剪接的组织、功能和细胞内位置 成分。 最直接需要回答的问题 重点关注哪些产品是结构组件 核酸内切酶。 一旦回答了这个核心问题，就应该 有可能解决更复杂的问题，例如如何以及在哪里 tRNA 剪接发生在完整细胞中。