Factor-Dependent Splicing of Chloroplast Group II Introns

Barkan许多基本的生物学过程涉及结构复杂的大RNA分子，其折叠可能会出现问题。在细胞中，结构RNA与蛋白质形成复合体，或者需要与蛋白质进行短暂的相互作用才能有效发挥作用。这种相互作用可能有助于增强正确折叠的RNA的形成或稳定性。自剪接的I组和II组内含子是高度结构的催化分子，需要蛋白质在活体中有效地发挥作用。由于促进内含子功能的蛋白质可以从基因上鉴定并在体外进行研究，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。然而，对组II剪接因子的研究相对滞后。该项目涉及玉米中的两个核基因，这两个基因是体内剪接特定叶绿体II组内含子所必需的。这些基因提供了独特的工具来解决蛋白质在促进第二组内剪接中的作用。Crs1基因是剪接thepF内含子所必需的；CRS2基因是剪接叶绿体中10个亚组IIB内含子中的9个所必需的。该项目目前的目标是确定每种蛋白质在剪接过程中所扮演的角色。最近利用转座子标签技术克隆了crs1和crs2基因。CRS1是一种新型的亲水性蛋白质。CRS2与肽基-tRNA水解酶有关，它的功能是回收以流产翻译产物形式释放的肽基-tRNA。现在将使用体外和“在大肠杆菌中”的RNA结合和剪接试验来区分这些蛋白质在剪接过程中所扮演的角色的几种模型。在体外或体内，只有少数蛋白质被证明能增强催化RNA的功能。要知道这种相互作用的机制库将有多大，这是很难的。因此，这些实验的结果可能有助于对RNA折叠问题和蛋白质在RNA催化反应中的作用的基本见解。这些结果还将阐明高等植物核和叶绿体基因组之间调控相互作用的本质。摘要：细胞内发生的化学反应是由称为酶的分子催化的，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，但也有一些是由RNA组成的，这与DNA相似，DNA是遗传信息的载体。不管它的化学组成如何，酶的折叠成正确的三维形状对它的功能至关重要。蛋白质折叠的机制多年来一直被研究。从这项工作中得出的一个基本概念是，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，这些分子被称为“伴侣”。关于催化的RNA分子如何呈现其正确的结构，我们知道得很多。这个项目涉及两种蛋白质，CRS1和CRS2，它们是某些催化的RNA分子发挥功能所必需的。这个项目的目标是了解这些蛋白质在促进RNA催化的反应中所起的作用。特别是，这个项目探索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能增强催化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.