Regulation of Photosynthetic Electron Transport Genes in Chlamydomonas Chloroplasts

光合作用中的电子传递主要由多亚基复合物完成，其成分由核和叶绿体基因编码。这些复合物的合成依赖于这两个细胞器中协调的基因表达。这种相互依赖的一个特别有趣的方面是，许多这些核基因调节特定叶绿体基因产物的合成。这种调控主要在转录后水平进行，介导RNA加工、RNA稳定性、翻译和大分子复合物组装等过程。本研究将重点关注单细胞绿藻莱茵衣藻叶绿体中的核调控基因及其RNA靶点，特别是那些编码电子传递链细胞色素b6/f复合物亚基的基因。细胞色素b6/f复合物介导质体醌池和质体青素之间的电子传递，并受发育和环境调控。该项目的具体目标是首先，分析两种破坏叶绿体petA和petD mrna稳定性的突变，它们分别编码细胞色素f和亚基IV。其次，叶绿体核糖体在mRNA中的识别元件将被功能定义。核基因产物和控制mRNA稳定性的机制分析将同时利用分子遗传学和生物化学进行。分子方法将用于研究mRNA稳定性突变中降解发生的机制，并克隆由一个这样的突变鉴定的基因。生化方法将利用体外系统来分析RNA-蛋白质相互作用的细节，重点关注这些稳定因子识别特定RNA序列的能力，以及可能执行其他功能，如RNA加工。我们假设IV亚基mRNA稳定因子编码mRNA 5'非翻译区结合蛋白，利用该位点的分子标记等位基因克隆编码该蛋白的核基因将有助于分析该亚基mRNA稳定因子。为了分析核糖体识别元件，将测试靠近翻译起始密码子的两个mRNA区域在核糖体RNA识别中的可能作用。其中一个区域是Shine-Dalgarno元件，其在叶绿体mrna中的存在尚未确定。另一个区域由起始密码子上游的三个核苷酸组成。这些元素的突变，以及核糖体RNA中潜在的配对伙伴。将用于测试这两种RNA分子的相互作用。这项研究的总体目标是促进我们对叶绿体基因调控的理解，以一种可能广泛适用于光合真核生物的方式。一些结果可能阐明了细胞核用来控制细胞质细胞器的进化策略。光合作用下的电子传递主要由多亚基复合物完成，其成分由核和叶绿体基因编码。这些复合物的合成依赖于这两个细胞器中协调的基因表达。这种相互依赖的一个特别有趣的方面是，这些核基因中的许多都是调控和控制特定叶绿体基因产物的合成。因此，曾经在该细胞器的原核祖先中自主表达的基因现在需要特定的核基因产物才能发挥活性。本文主要研究了单细胞绿藻莱茵Chamydomonas reinhardtii叶绿体中的核调控基因及其RNA靶点。该研究将有助于我们对叶绿体基因调控的理解，在某种程度上可能广泛适用于光合真核生物。一些结果可能阐明了细胞核用来控制细胞质细胞器的进化策略。＊＊＊

9723274 Stern Photosynthetic electron transport is largely carried out by multi-subunit complexes, whose constituents are encoded by nuclear and chloroplast genes. The synthesis of these complexes relies on the coordinate gene expression in these two organelles. A particularly intriguing aspect of this interdependence is that many of these nuclear genes regulate the synthesis of a specific chloroplast gene product. This regulation is carried out largely at the post-transcriptional level, mediating processes such as RNA processing, RNA stability, translation, and macromolecular complex assembly. This research will focus on nuclear regulatory genes and their RNA targets in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii, particularly those that encode subunits of the cytochrome b6/f complex of the electron transport chain. The cytochrome b6/f complex mediates electron transfer between the plastoquinone pool and plastocyanin, and is subject to developmental and environmental regulation. The specific goals of this project are first, to analyze two mutations that impair the stability of the chloroplast petA and petD mRNAs, which encode cytochrome f and subunit IV, respectively. Second, chloroplast ribosome recognition elements in mRNA will be functionally defined. The analysis of nuclear gene products and mechanisms that control mRNA stability will be carried out simultaneously using molecular genetics and biochemistry. Molecular approaches will be used to investigate the mechanism by which degradation occurs in mRNA stability mutants, and to clone the gene identified by one such mutation. Biochemical approaches will take advantage of in vitro systems to analyze the details of RNA-protein interactions, focusing on the ability of these stability factors to recognize specific RNA sequences, and perhaps to carry out other functions, such as RNA processing. The analysis of the subunit IV mRNA stability factor, which we hypothesize to encode an mRNA 5' untranslated reg ion-binding protein, will be facilitated by using a molecularly tagged allele of this locus to clone the nuclear gene encoding this protein. To analyze ribosome recognition elements, two regions of mRNA close to the translation initiation codon will be tested for a possible role in ribosomal RNA recognition. One of these regions is a Shine-Dalgarno element whose existence in chloroplast mRNAs is not yet firmly established. The other region consists of the three nucleotides immediately upstream of the initiation codon. Mutagenesis of these elements, and potential pairing partners in ribosomal RNA, .will be used to test the proposed interaction of these two RNA molecules. The overall goal of this research is to contribute to our understanding of chloroplast gene regulation, in a way that may be broadly applicable to photosynthetic eukaryotes. Some of the results may illuminate evolutionary strategies used by the nucleus to seize control of this cytoplasmic organelle. %%% Photosynthetic electron transport is largely carried out by multi-subunit complexes, whose constituents are encoded by nuclear and chloroplast genes. The synthesis of these complexes relies on the coordinate gene expression in these two organelles. A particularly intriguing aspect of this interdependence is that many of these nuclear genes are regulatory and control the synthesis of a specific chloroplast gene product. Therefore, genes once expressed autonomously in the prokaryotic ancestor of this organelle now require specific nuclear gene products for their activity. This research focuses on nuclear regulatory genes and their RNA targets in the chloroplast of the unicellular green alga Chamydomonas reinhardtii. The research will contribute to our understanding of chloroplast gene regulation, in a way that may be broadly applicable to phosynthetic eukaryotes. Some of the results may illuminate evolutionary strategies used by the nucleus to seize control of this cytoplasmic organelle. ***