Chloroplast RNA-binding proteins involved in RNA processing and translation

(1)叶绿体制备条件考虑到豌豆适合生物化学研究，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的30个mrna在其5'-UTRs中没有Shine-Dalgarno-like序列，需要反式因子进行翻译。利用我们的叶绿体体外翻译系统，我们确定了用于翻译的顺式元件。利用我们的叶绿体体外翻译系统，我们鉴定了叶绿体atpB mRNA翻译的顺式元件。烟草叶绿体rps2 mRNA具有一个Shine-Dalgarno-like序列，但令人惊讶的是，该序列被发现具有翻译抑制作用。(3)叶绿体RNA编辑相关因素分析为了分析叶绿体RNA编辑的分子机制，我们首次建立了叶绿体体外系统。利用该系统，我们确定了psbL mRNA和psbE mRNA的位点识别因子，分别为25 kDa和56 kDa蛋白。我们试图分离出这些蛋白质，但到目前为止还没有成功。(4)豌豆叶绿体的RNA编辑分析作为第二种模式系统，我们利用分离的豌豆叶绿体建立了离体系统。使用该系统，我们发现由于豌豆失去了其编辑位点，psbE mRNA没有编辑活性。这表明编辑场所和编辑活动是共同进化的。

(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.