Chloroplast RNA-binding proteins involved in RNA processing and translation

（1）叶绿体分离条件由于豌豆适合生化研究，我们对其生长条件、叶片收获和分离叶绿体的制备进行了优化。然后，我们在豌豆叶绿体转录本中搜索RNA编辑位点。（2）叶绿体翻译的顺式和反式因子分析烟草叶绿体中的30种mRNA在其5 ′-UTR中没有Shine-Dalgarno-like序列，需要反式因子进行翻译。使用我们的叶绿体在体外翻译系统，我们确定了翻译顺式元件。利用我们的叶绿体体外翻译系统，我们确定了叶绿体atpB mRNA翻译的顺式元件。烟草叶绿体rps 2 mRNA具有Shine-Dalgarno样序列，但令人惊讶的是，发现该序列抑制翻译。（3）叶绿体RNA编辑的影响因素分析为了研究叶绿体RNA编辑的分子机制，我们首次建立了叶绿体RNA编辑的体外系统。使用这个系统，我们确定了psbL mRNA和psbE mRNA，这是25 kDa和56 kDa的蛋白质，分别位点识别因子。我们试图分离这些蛋白质，但迄今为止没有成功。（4）豌豆叶绿体中的RNA编辑分析作为第二个模型系统，我们从分离的豌豆叶绿体中开发了一个叶绿体体外系统。使用这个系统，我们发现没有编辑活性的psbE mRNA的豌豆失去了它的编辑位点。这表明编辑活动和编辑场所是共同进化的。

(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.