TRNA SPLICING ENDONUCLEASE IN YEAST

从Btensor-tRNA剪接插入序列需要 至少两种功能，核酸内切酶和连接酶。 这些 酶催化内含子的切除和 tRNA半分子通过识别的保守特征， tRNA结构 尽管tRNA剪接很容易 我们对基因和生化分析的结果知之甚少 关于相关的调节和结构基因， 蛋白质或其他成分的相互作用， 剪接复合体，或剪接和 tRNA从细胞核到细胞质的转运。 信息 tRNA剪接的这些方面还没有出现， 膜相关tRNA剪接的难处理性 核酸内切酶。 之前几个实验室尝试研究 通过正向或反向遗传技术 都没有成功。 然而，最近，SEN中的突变 （剪接核酸内切酶）基因在本实验室鉴定 其导致在细胞中核酸内切酶活性的部分缺陷， 酿酒酵母 一个突变体也会导致前- tRNA稳态积累和温度敏感性 增长 通过使用遗传学方法分析SEN基因及其产物， 生物化学和免疫学的研究方法， 建议书将提供有关结构的重要信息， tRNA剪接的组织、功能和细胞内定位 件. 最迫切需要回答的问题是 中心，产品是 核酸内切酶。 一旦这个核心问题得到回答， 如何以及在哪里解决更复杂的问题 tRNA剪接发生在完整的细胞中。

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.