Gene-specific and General RNA Regulators in Chloroplasts

叶绿体基因的表达是光合作用所必需的，并且必须与核基因组相协调。这种协调是由一套主要是转录后调控因子实现的，其中一些具有可识别的原核来源，另一些似乎是在内生共生后独特获得的。一个引人入胜且在很大程度上尚未解决的问题是，这些来源迥异的蛋白质如何相互作用，影响基因特异性和一般控制叶绿体基因的表达。本项目以绿藻衣藻为模型，探索MCD1和MCD4两种调控蛋白之间的关系。MCD1编码一个大的、新的蛋白质，它似乎是多聚体复合体的成员，并且是积累叶绿体PETD mRNA所必需的，它通过与PETD 5‘非翻译区的相互作用来保护叶绿体免受核糖核溶解的衰退。在携带PETD 5‘UTR突变的菌株的抑制子筛选中分离到了mcd4，这些突变破坏了转录本的稳定性，然而mcd4抑制子被证明是多效性的，在其他叶绿体转录本中显示出许多缺陷。这项研究的具体目的是：(1)通过体外RNA结合分析来探索MCD1蛋白的生化活性，以确定它是否具有解释PETD专一性的特性；(2)纯化含有MCD1的多蛋白复合体，并确定其成分；(3)鉴定多效性基因MCD4；以及(4)利用遗传杂交，特别是它可能调节的两种候选核糖核酸酶，即多核苷酸磷酸化酶和RNase J1，研究MCD4与其他RNA代谢因子的特异性和相互作用。叶绿体是进行光合作用的地方，在古代被捕获为细菌。在这个项目中，位于细胞核中的两个基因正在研究中，因为它们调节最初与细菌遗传的叶绿体基因，这是光合作用所必需的。这项研究将为未来的科学家提供机会，无论是暑期实习生还是大学本科生，都可以获得宝贵的经验，品尝生物学研究的兴奋。过去几年，在这些实习生中，代表性不足的群体一直很突出。该项目的负责人还参与了通过广播节目MicrobeWorld、科学歌舞厅和其他工具对普通公众进行植物科学教育。

Chloroplast gene expression is essential for photosynthesis, and has to be coordinated with the nuclear genome. This coordination is achieved by a suite of mostly post-transcriptional regulators, some of which have recognizable prokaryotic origins, and others of which appear to have been uniquely derived following endosymbiosis. A fascinating and largely unresolved question is how these proteins of very different origins interact to effect both gene-specific and general control of chloroplast gene expression. This project uses the green alga Chlamydomonas reinhardtii as a model to explore the relationship between two regulatory proteins, MCD1 and MCD4. MCD1 encodes a large, novel protein which appears to be a member of a multimeric complex, and is specifically required for accumulation of the chloroplast petD mRNA, which it protects from ribonucleolytic decay through interaction with the petD 5' untranslated region. MCD4 was isolated in a suppressor screen of strains carrying petD 5' UTR mutations which destabilize the transcript, however the mcd4 suppressor proved to be pleiotropic, displaying numerous defects in other chloroplast transcripts. The specific aims of this research are to: (1) explore the biochemical activities of the MCD1 protein through in vitro RNA-binding assays, to determine if it has properties which can account for its specificity for petD; (2) purify the multiprotein complex containing MCD1, and determine its components; (3) identify the pleiotropic gene MCD4; and (4) study the specificity and interactions of MCD4 with other RNA metabolic factors using genetic crosses, in particular two candidate ribonucleases which it might regulate, polynucleotide phosphorylase and RNase J1.This project explores an example of how plant cells have evolved to coordinate activities in different compartments. The chloroplast, where photosynthesis occurs, was captured in ancient times as a bacterium. In this project, two genes located in the nucleus are under study, since they regulate chloroplast genes, originally inherited with the bacterium, which are required for photosynthesis. The research will offer opportunities for future scientists, either as summer interns or university undergraduates, to gain valuable experience and taste the excitement of biological research. Underrepresented groups have been prominent among these interns in past years. The leader of this project is also involved in educating the lay public about plant science, through the radio program MicrobeWorld, a Science Cabaret, and other vehicles.