Factor-Dependent Splicing of Chloroplast Group II Introns

许多重要的生物过程都涉及结构复杂的大型RNA分子，这些分子的折叠很可能存在问题。在细胞中，结构rna与蛋白质复合物或需要与蛋白质的短暂相互作用才能有效地发挥作用。这种相互作用可能有助于增强正确折叠RNA的形成或稳定性。“自剪接”I族和II族内含子是高度结构化的催化分子，需要蛋白质在体内有效地发挥作用。因为促进内含子功能的蛋白质可以通过基因鉴定，然后在体外进行研究，这为探索蛋白质在RNA催化反应和RNA折叠中的作用提供了方便的模型。促进真菌线粒体中I组内含子剪接的两个因素已被详细研究。然而，对II族剪接因子的研究相对滞后。本项目涉及玉米体内特定叶绿体II族内含子剪接所需的两个核基因。这些基因为解决促进II组内含子剪接的蛋白质角色提供了独特的工具。crs1基因是theatpF内含子剪接所必需的；crs2基因是叶绿体中10个IIB亚群内含子中9个的剪接所必需的。该项目的当前目标是确定每种蛋白质在剪接过程中所起的作用。crs1和crs2基因是最近通过转座子标记克隆的。CRS1是一种新型的亲水性蛋白。CRS2与肽基trna水解酶有关，肽基trna水解酶的功能是回收作为失败翻译产物释放的肽基trna。体外和“大肠杆菌内”RNA结合和剪接试验现在将用于区分这些蛋白质在剪接过程中的作用的几种模型。在体外或体内，只有少数蛋白质被证明可以增强催化rna的功能。要知道这种相互作用的机制有多大还为时过早。因此，这些实验的结果可能会导致对RNA折叠问题和蛋白质在RNA催化反应中的作用的基本见解。研究结果还将阐明高等植物核与叶绿体基因组之间调控相互作用的性质。概述：细胞内发生的化学反应是由一种叫做酶的分子催化的，这种分子在生理条件下促进化学键的断裂和形成。大多数酶是由蛋白质组成的，但有些是由RNA组成的，RNA类似于DNA，是遗传信息的载体。不管它的化学组成如何，将酶折叠成正确的三维形状对其功能至关重要。蛋白质折叠的机制已经被研究了很多年。从这项工作中产生的一个基本概念是，大多数蛋白质不能自发折叠成正确的形状，而是需要其他蛋白质分子的帮助，这些蛋白质分子被称为“伴侣”。对于催化RNA分子是如何形成其正确结构的，我们所知甚少。该项目涉及两种蛋白质，CRS1和CRS2，它们是某些催化RNA分子功能所必需的。本项目的目标是了解这些蛋白质在促进rna催化反应中所起的作用。特别是，该项目探讨了这些蛋白质如何调节催化RNA分子的折叠或稳定性。只有少数蛋白质被证明可以增强催化rna的功能。因此，这些实验的结果可能会导致对RNA折叠问题和蛋白质在RNA催化反应中的作用的基本见解。

BarkanMany essential biological processes involve large, intricatelystructured RNA molecules whose folding is likely to be problematic. In thecell, structured RNAs are complexed with proteins or require transientinteractions with proteins to function efficiently. Such interactions mayserve to enhance the formation or stability of the correctly folded RNA."Self-splicing" group I and group II introns are highly structuredcatalytic molecules that require proteins to function efficiently in vivo.Because proteins that facilitate intron function can be identifiedgenetically and then studied in vitro, these provide convenient models forprobing the role of proteins in RNA catalyzed reactions and RNA folding.Two factors that facilitate the splicing of group I introns in fungalmitochondria have been examined in great detail. However, the study ofgroup II splicing factors has lagged behind. This project concerns twonuclear genes in maize that are required in vivo for the splicing ofspecific chloroplast group II introns. These genes provide unique toolsfor addressing the roles of proteins in facilitating group II intronsplicing. The crs1 gene is required specifically for the splicing of theatpF intron; the crs2 gene is required specifically for the splicing ofnine of the ten subgroup IIB introns in the chloroplast. The current goalof this project is to define the role played by each protein in thesplicing process. The crs1 and crs2 genes were recently cloned bytransposon tagging. CRS1 is a novel, hydrophilic protein. CRS2 isrelated to peptidyl-tRNA hydrolases, which function to recyclepeptidyl-tRNAs that are released as abortive translation products. Invitro and "in E. coli " RNA binding and splicing assays will now be usedto distinguish between several models for the roles of these proteins inthe splicing process. Only a handful of proteins have been shown toenhance the function of catalytic RNAs in vitro or in vivo. It is toosoon to know how large the repertoire of mechanisms will be for suchinteractions. Therefore, the results of these experiments are likely tolead to fundamental insights into the RNA folding problem and the role ofproteins in RNA catalyzed reactions. The results will also elucidate thenature of the regulatory interactions between the nuclear and chloroplastgenome in a higher plant.Lay summary: The chemical reactions that occur inside cells are catalyzed bymolecules called enzymes that facilitate the breaking and forming ofchemical bonds under physiological conditions. Most enzymes are composedof protein but some are composed of RNA, which is similar to DNA, thecarrier of genetic information. Regardless of its chemical makeup, thefolding of the enzyme into its correct three dimensional shape is criticalfor its function. Mechanisms by which proteins fold have been studied formany years. One fundamental concept to arise from this work is that mostproteins cannot fold spontaneously into their correct shape, but ratherrequire help from other protein molecules, dubbed "chaperones". Much lessis known about how catalytic RNA molecules assume their correct structure.This project concerns two proteins, CRS1 and CRS2, that are required forthe function of certain catalytic RNA molecules. The goal of this projectis to come to an understanding of the role these proteins play infacilitating the RNA-catalyzed reactions. In particular, this projectexplores how these proteins modulate the folding or stability of thecatalytic RNA molecules. Only a handful of proteins have been shown toenhance the function of catalytic RNAs. Therefore, the results of theseexperiments are likely to lead to fundamental insights into the RNA folding problem and the role of proteins in RNA catalyzed reactions.