REGULATION OF CHLOROPHYLL/APOPROTEIN SYNTHESIS

叶绿素/脱辅蛋白合成的调节

• 批准号: 2179073
• 负责人: JOHN E. MULLET
• 金额: $ 16.86万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179073
• 关键词: Arabidopsis; RNA binding protein; biological signal transduction; chemical binding; chlorophyll; chloroplasts; genetic promoter element; genetic translation; genetically modified plants; laboratory rabbit; membrane proteins; molecular cloning; phosphorylation; photosystem; plant proteins; protein biosynthesis; tobacco; transcription factor

The long range goal of this research is to elucidate biochemical mechanisms which regulate the synthesis of chlorophyll-protein in chloroplasts of higher plants. The study of chlorophyll-protein synthesis provides special insight into the general process of membrane protein synthesis and cofactor assembly. This is because chlorophyll is highly fluorescent and provides a sensitive probe into the assembly process. Furthermore, because chlorophyll biosynthesis is light dependent in higher plants, the process can be easily controlled. The crystal structure of bacterial reaction centers containing the subunits L and M have been obtained. Bacterial reaction centers and l and M, are homologous to higher plant photosystem II reaction centers and their subunits, D1 and D2. Therefore, analysis of reaction center chlorophyll-protein synthesis has a strong biophysical and structural basis. Thr proposed research will focus ont he synthesis of D1 and D2, two membrane-bound, chlorophyll-binding proteins which form the reaction center of Photosystem II. D1 and D2 are encoded by the plastid genes psbA and psbD. The accumulation of these proteins is regulated through modulation of RNA stability, translation initiation, translation elongation (ribosome pausing), cofactor binding and activation of a special blue light inducible promoter. Three proteins, RNP95, RNP48 and RNP60, have been identified which bind to the psbA 5'-untranslated RNA. A specific binding site for RBNP95 has been defined. Regulation of psbA mRNA stability and translation initiation may occur through the action of these RNA binding proteins. The gene encoding RNP95 will be cloned and used to examine the role of RNP95 in modulating psbA mRNA stability and translation regulation in response to light intensity, light/dark cycles and chloroplast development. Plastid translation is also regulated at the level of translation elongation during light/dark cycles. This general regulation of elongation may modulate the amount of selective ribosome pausing observed during translation of the chlorophyll-protein D1. The mechanistic basis of light modulated translation elongation will be investigated by extract complementation and translation elongation assays. In addition, the relationship between ribosome pausing, chlorophyll binding, and stabilization of paused chlorophyll-apoprotein translation intermediates will be investigated. The chlorophyll-proteins D1 and D2 are damaged and turnover as a consequence of photochemistry especially at high light. In response, plants activate transcription from a blue light inducible promoter which transcribes psbD, the gene encoding D2. The biochemical mechanisms which regulates the blue light inducible psbD promoter will be investigated. Promoter constructs and DNA binding protein function will be analyzed using an in vitro transcription system. The role of reversible phosphorylation of DNA binding proteins and the blue light signal transduction pathway will be characterized.

这项研究的长期目标是阐明生化机制。 它们调节黄瓜叶绿体中叶绿素蛋白的合成 高等植物。对叶绿素-蛋白质合成的研究提供了特殊的 洞察膜蛋白合成的一般过程及其辅因子 集合。这是因为叶绿素是高度荧光的，并提供 对组装过程的敏感探查。此外，因为 在高等植物中，叶绿素的生物合成是依赖于光的，这个过程 可以很容易地控制。细菌反应的晶体结构 得到了含有L和M亚基的中心。细菌 反应中心和L、M，与高等植物光系统II同源 反应中心及其亚基，d1和d2。因此，分析 反应中心的叶绿素-蛋白质合成具有很强的生物物理和 结构性基础。建议的研究将集中在D_1的合成上。 和D2，两种膜结合的叶绿素结合蛋白，形成了 光系统II的反应中心D_1和D_2由叶绿体编码 PSBA和PSBD基因。这些蛋白质的积累是受调控的。 通过调节RNA的稳定性、翻译起始、翻译 延长(核糖体暂停)、辅因子结合和激活一种特殊的 蓝光诱导启动子。RNP95、RNP48和RNP60三种蛋白质， 已鉴定出与PSBA 5‘-未翻译的RNA结合的RNA。一个 已经确定了RBNP95的特异结合位点。PSBA基因的转录调控 稳定性和翻译启动可以通过这些因素的作用而发生 RNA结合蛋白。编码RNP95的基因将被克隆并用于 检测RNP95在调节PSBA mRNA稳定性和翻译中的作用 对光强度、明/暗周期和 叶绿体发育。叶绿体的翻译也在 在明/暗循环中的翻译伸长水平。这位将军 伸长的调节可能调节选择性核糖体的数量 在叶绿素蛋白D1的翻译过程中观察到停顿。这个 光调制平移伸长的机理基础将是 通过提取物互补和翻译延伸试验进行了研究。 此外，核糖体停顿、叶绿素 暂停的叶绿素-载脂蛋白翻译的结合和稳定 将对中间体进行调查。叶绿素蛋白d1和d2是 由于光化学造成的损坏和周转，特别是在高海拔地区 灯。作为回应，植物从蓝光中激活转录 可诱导启动子转录PbD，即编码D2的基因。这个 调控蓝光诱导PSBD的生化机制 将对发起人进行调查。启动子构建与DNA结合蛋白 功能将使用体外转录系统进行分析。角色 DNA结合蛋白的可逆磷酸化和蓝光 信号转导途径将被描述。