Ueda T, Tohda H, Chikazumi N, Eckstein F, Watanabe K
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Nucleic Acids Res. 1991 Feb 11;19(3):547-52. doi: 10.1093/nar/19.3.547.
Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase. RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.
使用核糖核苷5'-O-(1-硫代三磷酸)的Sp非对映体和T7 RNA聚合酶转录大肠杆菌噬菌体T7 DNA，生成含硫代磷酸酯的RNA。在由大肠杆菌和嗜热栖热菌制备的无细胞翻译系统中，单个核苷酸被相应的硫代磷酸酯核苷取代的RNA作为mRNA发挥作用。这种取代通过稳定这些系统中的mRNA提高了蛋白质合成效率。随着取代核苷酸比例的增加，它们的mRNA活性相应降低。根据SDS-聚丙烯酰胺凝胶电泳分析判断，以含硫代磷酸酯的mRNA为模板合成的蛋白质与用未取代的mRNA获得的蛋白质相同。然而，当使用硫代磷酸酯-RNA时，使用未取代的mRNA时几乎检测不到的较大蛋白质得到了很好的表达。讨论了在体外翻译系统中使用硫代磷酸酯-mRNA的优势。
