A sensitive in vitro editing assay for the pea chloroplast petB editing site has been developed and utilized to study the mechanism of C-to-U editing in chloroplast extracts. The in vitro editing assay was characterized by several criteria including: linearity with extract amount; linearity over time; dependence on assay components; and specificity of editing site conversion. The increase in the extent C-to-U conversion of the petB editing site was nearly linear with the amount chloroplast protein extract added, although the reaction appeared to decline in rate after approximately 30 min. The assay was tested for the importance of various assay components, and the omission of protease inhibitor and ATP was shown to dramatically reduce the extent of the editing reaction. Sequence analysis of cDNA clones obtained after an in vitro editing reaction demonstrated that 12 of 17 (71%) clones were edited, and that no other nucleotide changes in these cDNAs were detected. Thus, the fidelity and specificity of the in vitro editing system appears to be excellent, and this system should be suitable to study both mechanism of the editing reaction and editing site selection. The in vitro editing reaction was strongly stimulated by the addition of ATP, and all four NTPs and dNTPs stimulated the editing reaction except for rGTP, which had no effect. Thus, the nucleotide specificity of the editing reaction is broad, and is similar in this respect to the mitochondrial editing system. Most enzyme or processes specifically utilize ATP or GTP for phosphorylation and the ability to substitute other NTPs and dNTPs is unusual. RNA helicases have a similar broad nucleotide specificity and this may reflect the involvement of an RNA helicase in plant organelle editing.

中文翻译：

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豌豆叶绿体中RNA编辑反应的核苷酸特异性

豌豆叶绿体 petB 编辑位点的灵敏体外编辑测定已被开发并用于研究叶绿体提取物中 C-to-U 编辑的机制。体外编辑试验的特征有几个标准，包括：与提取物量的线性关系；随时间的线性；对化验成分的依赖；和编辑网站转换的特殊性。petB 编辑位点的 C 到 U 转换程度的增加与添加的叶绿体蛋白提取物的量几乎呈线性关系，尽管在大约 30 分钟后反应似乎速度下降。该测定针对各种测定成分的重要性进行了测试，并且证明省略蛋白酶抑制剂和 ATP 可显着降低编辑反应的程度。体外编辑反应后获得的 cDNA 克隆的序列分析表明，17 个 (71%) 克隆中有 12 个被编辑，并且未检测到这些 cDNA 中的其他核苷酸变化。因此，体外编辑系统的保真度和特异性似乎非常好，该系统应该适合研究编辑反应的机制和编辑位点选择。ATP 的加入强烈刺激了体外编辑反应，除 rGTP 没有影响外，所有四种 NTP 和 dNTP 都刺激了编辑反应。因此，编辑反应的核苷酸特异性是广泛的，在这方面与线粒体编辑系统相似。大多数酶或过程专门利用 ATP 或 GTP 进行磷酸化，替代其他 NTP 和 dNTP 的能力是不寻常的。