Site-recognition of RNA editing in chloroplasts

从高等植物叶绿体转录本的转录本中，在特定的位点发生C到U的RNA编辑。这种编辑在转录本中大约1,000个C中有一个C被观察到。为了分离大量的反式因子，设计了利用蔗糖梯度制备叶绿体的快速和经济的方法。德国同事给了我们很多有用的建议.体外RNA编辑系统的改进我们以前的体外系统需要β<32>-P标记的mRNA底物。由于合成β<32>-P标记的mRNA是繁琐和耗时的，我们使用引物延伸方法开发了具有荧光核苷酸的非RI体外系统。利用这个新的系统，我们检测了烟草叶绿体ndh mRNA的编辑活性。活动因地而异。30%（ndh 2）至不可检测。然后使用突变的mRNA鉴定ndh-2和ndhF mRNA编辑的顺式序列。ndh-2的顺式序列位于AUG上游10 - 6 nt，ndhF的顺式序列位于AUG上游40 - 36 nt和15 - 6 nt。由于ndhF mRNA具有两个顺式序列，我们提出了一种新的RNA编辑模型.由于psbE mRNA的编辑活性最高，因此，采用硫酸铵分级沉淀、肝素柱层析、凝胶过滤和SDS凝胶电泳的方法，从12 kg烟草绿色叶片中分离到了相应的56 kDa反式转录因子。切下56 kDa条带并进行MS/MS分析。获得了几个氨基酸序列。该蛋白可能是烟草特有的一个新蛋白，经BLAST检索未发现相似序列.使用UV交联，用于ndhF和rpoB mRNA编辑的反式因子分别被鉴定为82 kDa和60 kDa。

C to U RNA editing occurs in transcripts from higher plant chloroplast transcripts at specific sites. This editing is observed one C out of about 1,000 Cs in the transcripts.1. Improvement of chloroplast preparationTo isolate a sizable amount of trans-factors, rapid and economic procedure for preparing chloroplasts was devised using sucrose-gradients. Useful advices were come from our German colleagues.2. Improvement of the in vitro RNA editing systemOur previous in vitro system requires ^<32>P-labeled mRNA substrates. As the synthesis of ^<32>P-labeled mRNAs was tedious and time-consuming, we developed a non-RI in vitro system with fluorescent nucleotides using a primer extension method. Using the new system, we assayed editing activities for tobacco chloroplast ndh mRNAs. The activity varies from ca. 30% (ndh2) to not detectable. Cis-sequences for ndh-2 and ndhF mRNA editing were then identified using mutated mRNAs. The cis-sequence for ndh-2 resides 10 to 6 nt upstream from AUG and that for ndhF does 40 to 36 nt and 15 to 6 nt upstream from AUG. As ndhF mRNA has two cis-sequences, we proposed a new model for RNA editing.3. Isolation of a recognition factor for editingAs the highest editing activity was observed for psbE mRNA, the corresponding 56 kDa trans-factor was isolated from 12 kg tobacco green leaves using ammonium sulfate fractionation, heparin column, gel filtration and SDS gel electrophoresis. The 56 kDa band was excised and subjected to MS/MS analysis. Several amino acid sequences were obtained. This protein is likely to be a new protein unique to tobacco since no similar sequence was found by BLAST search.4. Identification of additional trans-factorsUsing UV crosslinking, trans-factors for ndhF and rpoB mRNA editing were identified to be 82 kDa and 60 kDa, respectively.