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

叶绿体是光合作用细菌的后代，因此包含它们自己的基因组和基因表达机制。然而，大多数叶绿体蛋白是核编码的，并在胞质核糖体上翻译，这就需要核和叶绿体基因产物的协调表达。质体基因的表达结合了原核生物的特征，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，如噬菌体类型转录、跨顺反子RNA加工、广泛的RNA剪接和编辑、转录特异性稳定剂和翻译激活剂。尽管叶绿体翻译和RNA稳定性的调节是叶绿体基因调控的主要方面，但人们对调节叶绿体翻译和RNA稳定性的因素，或翻译与蛋白质折叠、靶向和组装的耦合机制知之甚少。该项目将使用一种名为新生链多糖体免疫沉淀(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质的抗体，通过新生多肽链沉淀参与合成这些蛋白质的多聚体。用质谱仪分析免疫纯化的多聚体亚群的蛋白质组成，将确定翻译复合体中未知的转录/蛋白质特异性因子，这些因子可能在转录加工/稳定或翻译中起作用，以及蛋白质修饰、组装或靶向。通过突变和生化方法对选定的因子进行分析，将揭示它们在叶绿体基因表达中的功能。一个附带项目将描述具有异常叶绿体基因表达的玉米突变体，其潜在的突变已经被识别，但其机制尚不清楚。

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.