Suh E, Waring R B
Department of Biology, Temple University, Philadelphia, PA 19122.
Nucleic Acids Res. 1992 Dec 11;20(23):6303-9. doi: 10.1093/nar/20.23.6303.
RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions.
RNA聚合酶能够合成含有硫代磷酸酯键的RNA，其中硫取代了一个非桥连氧原子。转录过程中仅生成Rp异构体。嗜热四膜虫I组内含子5'剪接位点处的Rp硫代磷酸酯不抑制剪接（McSwiggen, J.A.和Cech, T.R.（1989年），《科学》244, 679）。在胞嘧啶或鸟苷三磷酸存在的情况下，将3'外显子的第一个碱基U+1分别突变为C或G的突变体进行转录时，会在3'剪接位点引入硫代磷酸酯。在这两种情况下，外显子连接均被阻断。在硫代磷酸酯取代的U+1G突变体中，在正确位点下游一个碱基处选择了一个新的3'剪接位点；尽管在未取代的RNA中以非常高的保真度选择了正确位点。相反，使用未取代的内含子RNA和在外显子连接位点含有Rp硫代磷酸酯的连接外显子成功地进行了反向外显子连接反应。正如在5'剪接位点切割中一样，转酯反应过程中手性发生了反转。这表明一个非桥连氧原子对这两种剪接反应都特别关键。
