SPLICING OF CELL ORGANELLE TRANSFER RNA PRECURSORS

这项研究的目的是确定生物化学机制 在高等植物叶绿体中的转移RNA剪接。 转移RNA基因 在其反密码子中具有0.4- 1.0kbp的内含子或间插序列， 已经描述了玉米、烟草、菠菜和豌豆叶绿体的区域 基因组 由于这些基因表达， 必须从初级转录物中剪接出来以产生功能性的tRNA。 本项目的具体目标是：（1）获得未剪接的前体 含内含子的叶绿体tRNA基因的转录本 转录，并通过RNA表征这些前体tRNA 指纹识别方法 (2)为准确拼接创造条件 这些前-tRNA，并表征干预的要求， 序列切除和外显子和内含子，以及剪接点。 （四） 为了部分纯化剪接所需的活性或因子， 体外并确定它们在剪接中的作用机制。 因此，为了确定拼接是否需要不同的因素， 不同的tRNA前体（例如，不同的核酸内切酶是否 位于反密码子不同位置的间插序列 循环）。 (5)为了确定这些内含子的二级结构， 开始研究内含子或tRNA内的结构是否 需要切除或结扎。 RNA剪接是真核细胞核表达的基本步骤， 和细胞器基因。 此外，叶绿体tRNA是一个理想的模型， 细胞器RNA剪接研究系统。 叶绿体tRNA 内含子可以表现出与提出的一类真菌的结构同源性， 植物线粒体mRNA内含子。 然而，每个前tRNA只有一个 单个内含子，并产生了一个最简单的生物化学系统， 一个细胞器内含子家族的代谢研究。 通过 比较了叶绿体tRNA剪接的机制与其它的剪接机制， 生物体，我们希望能够深入了解剪接机制的进化。

The goal of the proposed research is to determine the biochemical mechanism of transfer RNA splicing in higher plant chloroplasts. Transfer RNA genes with 0.4-1.0 kbp introns, or intervening sequences, in their anticodon regions have been described for maize, tobacco, spinach and pea chloroplast genomes. Since these genes are expressed, the intervening sequence RNA must be spliced out of the primary transcript to produce functional tRNAs. The specific aims of this project are: (1) To obtain unspliced precursor transcripts of intron-containing chloroplast tRNA genes by intro transcription, and to characterize these precursor tRNAs by RNA fingerprinting methods. (2) To establish conditions for accurate splicing of these pre-tRNAs, and to characterize the requirements for intervening sequence excision and for exon and intron, and of the splice junction. (4) To partially purify the activity(ies) or factor(s) required for splicing in vitro and to determine their mechanism of action in splicing. Consequently, to determine whether different factors are required to splice different tRNA precursors (e.g., whether different endonucleases excise intervening sequences located at different positions in the anticodon loop). (5) To determine the secondary structures of these introns and to begin to investigate whether structures within the intron or the tRNA are required for excision or ligation. RNA splicing is a fundamental step in the expression of eukaryotic nuclear and organnellar genes. Chloroplast tRNAs, moreover, are an ideal model system for studies on organellar RNA splicing. The chloroplast tRNA introns may exhibit structural homology with a proposed class of fungal and plant mitochondrial mRNA introns. Each pre-tRNA, however, has only a single intron, and gives rise to a simplest system for biochemical investigation of the metabolism of a family of organellar introns. By comparing the mechanism of chloroplast tRNA splicing with those of other organisms, we hope to gain insight to the evolution of splicing mechanisms.