TRNA SPLICING ENDONUCLEASE IN YEAST

从前体-tRNA中剪接插入序列需要 至少有两种功能，一种内切酶和一种连接酶。这些 酶催化内含子的切除和内含子的重新连接 TRNA半分子通过识别保守的特征 TRNA结构。尽管tRNA剪接很容易 服从于遗传和生化分析，人们知之甚少 关于涉及的调控和结构基因，潜在的 蛋白质或其他成分的相互作用，可能形成 剪接复合体，或者剪接和剪接之间的可能关系 TRNA从细胞核到细胞质的运输。有关的信息 TRNA剪接的这些方面还没有出现，原因是 膜相关tRNA剪接的棘手性质 核酸内切酶。之前在几个实验室进行的研究尝试 正向或反向基因技术的内切酶 都没有成功过。然而，最近，SEN的突变 (剪接内切酶)基因在本实验室被鉴定 这会导致部分核酸内切酶活性的缺陷 酿酒酵母。一个突变体也会导致前 TRNA的稳态积累和温度敏感性 成长。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 建议书将提供关于该结构的关键信息， TRNA剪接的组织、功能和细胞内定位 组件。最迫切需要回答的问题 以产品为结构组件的中心 核酸内切酶。一旦这个核心问题得到回答，它应该是 有可能解决更复杂的问题，如如何以及在哪里 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.