Discovery of proteins involved in the expression and assembly of specific chloroplast gene products through a novel polysome immunoprecipitation approach

通过新型多核糖体免疫沉淀方法发现参与特定叶绿体基因产物表达和组装的蛋白质

• 批准号: 199701000
• 负责人: Dr. Reimo Zoschke
• 金额: --
• 依托单位: Institute of Molecular Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/199701000?language=en
• 关键词: Discovery; proteins; involved; expression; assembly

Chloroplasts are the descendants of a photosynthetic bacterium, and thus contain their own genome and gene expression machinery. However, most chloroplast proteins are nuclear-encoded and translated on cytosolic ribosomes, necessitating coordinated expression of nuclear and chloroplast gene products. Plastid gene expression combines prokaryotic features such as polycistronic transcription units and eubacterial-like ribosomes, ribonucleases and RNA polymerase, with newly acquired, characteristics such as phage-type transcription, intercistronic RNA processing, extensive RNA splicing and editing, transcript specific stabilizers and translational activators. Those acquired features are performed by unusual families of RNA binding proteins whose members function primarily in organelles.Although the regulation of chloroplast translation and RNA stability are major aspects of chloroplast gene regulation, little is known about factors that adjust chloroplast translation and RNA stability, or about the mechanisms that couple translation to protein folding, targeting, and assembly. This project will screen for such factors by using an innovative approach named nascent-chain polysome immunoprecipitation (NCPIP). NCPIP uses antibodies against chloroplast-encoded proteins to precipitate polysomes engaged in the synthesis of these proteins via the nascent polypeptide chain. The analysis of the protein composition of immunopurified polysomal subsets by mass spectrometry will identify unknown transcript/protein specific factors of the translation complex, which could act in transcript processing/stability or translation, as well as protein modification, assembly or targeting. The analyses of selected factors by mutant and biochemical approaches shall reveal their functions in plastid gene expression. A side project will characterize maize mutants with aberrant chloroplast gene expression whose underlying mutations are already identified, but whose mechanisms are unknown.

叶绿体是光合细菌的后代，因此含有自己的基因组和基因表达机制。然而，大多数叶绿体蛋白质是核编码的，并在胞质核糖体上翻译，需要核和叶绿体基因产物的协调表达。质体基因表达结合了原核生物的特征，例如多顺反子转录单位和真细菌样核糖体、核糖核酸酶和RNA聚合酶，以及新获得的特征，例如噬菌体型转录、顺反子间RNA加工、广泛的RNA剪接和编辑、转录物特异性稳定剂和翻译激活剂。这些获得的特征是由不寻常的家族的RNA结合蛋白，其成员的功能主要在organelles.Although叶绿体翻译和RNA的稳定性的调节是叶绿体基因调控的主要方面，很少有人知道的因素，调节叶绿体翻译和RNA的稳定性，或有关的机制，耦合翻译蛋白质折叠，靶向和组装。该项目将通过使用一种名为新生链多核糖体免疫沉淀（NCPIP）的创新方法来筛选这些因素。NCPIP使用针对叶绿体编码蛋白的抗体来沉淀参与通过新生多肽链合成这些蛋白的多核糖体。通过质谱分析免疫纯化的多聚体亚群的蛋白质组成将鉴定翻译复合物的未知转录物/蛋白质特异性因子，其可在转录物加工/稳定性或翻译以及蛋白质修饰、组装或靶向中起作用。通过突变体和生物化学方法对选定的因子进行分析，将揭示它们在质体基因表达中的功能。一个附带项目将描述玉米叶绿体基因表达异常的突变体，其潜在的突变已经确定，但其机制尚不清楚。