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

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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.