REGULATION OF CHLOROPHYLL/APOPROTEIN SYNTHESIS

这项研究的长期目标是阐明生物化学机制 调节叶绿体中叶绿素蛋白的合成 高等植物 叶绿素蛋白质合成的研究提供了特殊的 了解膜蛋白合成和辅因子的一般过程 组装件. 这是因为叶绿素是高度荧光的， 对组装过程的敏感探测 此外，由于 在高等植物中，叶绿素的生物合成依赖于光， 可以很容易地控制。 细菌反应的晶体结构 已经获得了包含亚基L和M的中心。 细菌 反应中心l和M与高等植物光系统II同源 反应中心和它们的亚基，D1和D2。 因此分析 反应中心叶绿素蛋白质合成具有很强的生物物理和 结构基础。 本研究将集中于D1的合成 和D2，这两种膜结合的叶绿素结合蛋白， 光系统II的反应中心。 D1和D2由质体编码 psbA和psbD基因。 这些蛋白质的积累受到调节 通过调节RNA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个特殊的 蓝光诱导型启动子 三种蛋白质，RNP 95，RNP 48和RNP 60， 已经鉴定了与psbA 5 '-非翻译RNA结合的RNA。 一 RBNP 95的特异性结合位点已被确定。 psbA mRNA的调控 稳定性和翻译起始可以通过这些 RNA结合蛋白。 编码RNP 95的基因将被克隆并用于 研究RNP 95在调节psbA mRNA稳定性和翻译中的作用 调节响应于光强度、光/暗周期和 叶绿体发育 质体的翻译也受到调节， 在光/暗循环期间的平移伸长水平。 这个一般 延伸的调节可以调节选择性核糖体的量， 在叶绿素蛋白D1的翻译过程中观察到的停顿。 的 光调制平移伸长的机械基础将是 通过提取物互补和翻译延伸测定来研究。 此外，核糖体停顿，叶绿素 暂停叶绿素-脱辅基蛋白翻译的结合和稳定 将对中间体进行研究。 叶绿素蛋白D1和D2是 由于光化学作用，特别是在高温下， 光 作为回应，植物通过蓝光激活转录 诱导型启动子，其转录编码D2的基因psbD。 的 调节蓝光诱导psbD的生化机制 发起人将进行调查。 启动子构建体和DNA结合蛋白 将使用体外转录系统分析功能。 的作用 DNA结合蛋白的可逆磷酸化和蓝光 将表征信号转导途径。

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.