ORGANIZATION & EXPRESSION OF CHLOROPLAST TRNA GENES

叶绿体基因组中的许多转运RNA基因已经被发现， 被发现编码在多顺反子操纵子中。 这些操纵子可以 含有其他tRNA、rRNA的基因， 蛋白质，或其表达在发育过程中 监管. 大多数植物叶绿体基因表达的调控 基因发生在转录后。 监管机制 这可能涉及RNA加工事件和/或翻译 引发/延伸步骤是未知的。 的重点 建议是确定RNA成熟途径， 发育调节和组成型表达的叶绿体 多顺反子操纵子，并确定RNa加工的作用 步骤，在任何情况下，调节细胞器的发展。 有所侧重 将被放置在混合的tRNA-mRNA操纵子上，因为前体RNA 积累和tRNA加工必须进行mRNA翻译。 的 研究的主要目的是：（1）确定 多顺反子前体转录物的一级结构 操纵子;（2）确定前体转录物和非- 在前质体中积累的翻译mRNA位于 多核糖体或核糖核蛋白颗粒;（3）表征 tRNA-mRNA混合叶绿体操纵子的RNa加工途径 含有组成型表达的基因和光合作用 膜基因;（4）确定是否有发育 细胞器生物发生过程中的特定RNA加工事件;（5） 纯化选定的RNA加工酶，并确定是否RNa 发展调节;（6）表征一类新的 叶绿体内含子只存在于组成型表达基因中。 为了表征细胞器RNA，我们正在开发一种 制备叶绿体转录物的cDNA克隆的方法。 该程序将适用于成熟RNA种类，初级 转录物和RNa加工中间体。 该程序将 对任何来源的成绩单都是通用的，不限于 叶绿体RNA，并将适用于多顺反子 转录物，包括混合的tRNA-mRNA操纵子。 这些cDNA将 随后用T3或T7病毒调节剂在体外表达 序列和启动子作为一种手段，以产生底物， RNa加工反应的体外表征。

Many of the transfer RNA genes of chloroplast genomes have been found to be encoded in polycistronic operons. These operons can contain genes for other tRNAs, rRNAs, constitutively expressed proteins, or proteins whose expression is developmentally regulated. Regulation of chloroplast gene expression for most genes occurs posttranscriptionally. The regulatory mechanisms which could involve RNA processing events and/or translation initiation/elongation steps are not known. The focus of this proposal is to determine the RNA maturation pathways for developmentally regulated and constitutively expressed chloroplast polycistronic operons, and to determine the role of RNa processing steps, in any, in regulating organelle development. Some emphasis will be placed on mixed tRNA-mRNA operons, since precursor RNAs accumulate and tRNA processing must proceed mRNA translation. The main objectives of the proposed research are: (1) to determine the primary structure of precursor transcripts from polycistronic operons; (2) to determine if precursor transcripts and non- translated mRNAs that accumulate in proplastids are localized on polysomes or in ribonucleoprotein particles; (3) to characterize the RNa processing pathways for tRNA-mRNA mixed chloroplast operons containing constitutively expressed genes and photosynthetic membrane genes; (4) to determine if there are developmental specific RNA processing event during organelle biogenesis; (5) to purify selected RNA processing enzymes, and determine if the RNa developmentally regulated; (6) to characterize a novel class of chloroplast introns found only in constitutively expressed genes. In order to characterize organelle RNAs, we are developing a procedure for preparing cDNA clones of chloroplast transcripts. The procedure will be applicable to mature RNA species, primary transcripts, and RNa processing intermediates. The procedure will be general for transcripts from any source, not restricted to chloroplast RNAs, and will be applicable to polycistronic transcripts, including mixed tRNA-mRNA operons. These cDNAs will be subsequently expressed in vitro with T3 or T7 viral regulatory sequences and promoters as a means to generate substrates for in vitro characterization of RNa processing reactions.