Regulation of Photosynthetic Electron Transport Genes in Chlamydomonas Chloroplasts

衣藻叶绿体中光合电子传递基因的调控

• 批准号: 9723274
• 负责人: David Stern
• 金额: $ 27万
• 依托单位: Boyce Thompson Institute Plant Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723274&HistoricalAwards=false
• 关键词: Regulation; Photosynthetic; Electron; Transport; Genes

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. ***

9723274 Stern光合电子传递主要由多亚基复合体进行，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种细胞器中基因的协调表达。 这种相互依赖性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。 这种调节主要在转录后水平进行，介导诸如RNA加工、RNA稳定性、翻译和大分子复合物组装的过程。 本研究将集中在核调控基因和它们的RNA靶在单细胞绿色衣藻的叶绿体，特别是那些编码亚基的细胞色素b6/f复合物的电子传递链。细胞色素b6/f复合物介导质体醌库和质体蓝素之间的电子转移，并受到发育和环境调节。 该项目的具体目标是首先分析两个突变，这两个突变损害叶绿体petA和petD mRNA的稳定性，分别编码细胞色素f和亚基IV。 第二，叶绿体核糖体识别元件的mRNA将功能定义。 核基因产物和控制mRNA稳定性的机制的分析将同时使用分子遗传学和生物化学进行。 分子方法将被用来研究mRNA稳定性突变体中发生降解的机制，并克隆由一个这样的突变鉴定的基因。 生物化学方法将利用体外系统来分析RNA-蛋白质相互作用的细节，重点是这些稳定性因子识别特定RNA序列的能力，以及可能执行其他功能的能力，例如RNA加工。 亚基IV mRNA稳定因子的分析，我们假设编码mRNA 5'非翻译区结合蛋白，将通过使用该位点的分子标记等位基因克隆编码该蛋白的核基因来促进。 为了分析核糖体识别元件，将测试靠近翻译起始密码子的mRNA的两个区域在核糖体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。 另一个区域由紧邻起始密码子上游的三个核苷酸组成。 这些元件的突变，以及核糖体RNA中潜在的配对伴侣，将用于测试这两种RNA分子的拟议相互作用。 这项研究的总体目标是有助于我们了解叶绿体基因调控，在某种程度上，可能是广泛适用于光合真核生物。 一些结果可能会阐明细胞核控制细胞质细胞器的进化策略。 光合电子传递主要由多亚基复合体完成，其组分由核和叶绿体基因编码。 这些复合物的合成依赖于这两种细胞器中基因的协调表达。 这种相互依赖性的一个特别有趣的方面是，许多这些核基因是调节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来进行活性。 本研究以单细胞绿色衣藻（Chamyspacereinhardtii）叶绿体中的核调控基因及其RNA靶基因为研究对象。 这项研究将有助于我们了解叶绿体基因调控，在某种程度上，可能广泛适用于光合真核生物。 一些结果可能会阐明细胞核控制细胞质细胞器的进化策略。 ***