Chloroplast RNA-binding proteins involved in RNA processing and translation

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(1)Conditions for chloroplast preparationAs pea is suitable for biochemical studies, we optimized its growth conditions, leaf harvest and preparation of isolated of isolated chloroplasts. Then, we searched for RNA editing sites in pea chloroplast transcripts.(2)Analysis of cis-and trans-factors for chloroplast translationThirty mRNAs from tobacco chloroplasts have no Shine-Dalgarno-like sequences in their 5'-UTRs and require trans-factors for translation. Using our chloroplast in vitro translation system, we identified cis-elements for translation. Using our chloroplast in vitro translation system, we identified cis-elements for translation of chloroplast atpB mRNA. The tobacco chloroplast rps2 mRNA possesses a Shine-Dalgarno-like sequence, but surprisingly this sequence was found to be inhibitory for translation.(3)Analysis of factors involved in chloroplast RNA editingTo analyze the molecular mechanism of RNA editing in chloroplasts, we developed for the first time a chloroplast in vitro system. Using this system, we identified site-recognition factors for psbL mRNA and psbE mRNA, which are 25 kDa and 56 kDa proteins, respectively. We tried to isolate these proteins but so far unsuccessful.(4)RNA editing analysis in pea chloroplastsAs a second model system, we developed a chloroplast in vitro system from isolated pea chloroplasts. Using this system, we found no editing activity for psbE mRNA as pea lost its editing site. This indicates that editing sites and editing activities are co-evolued.