Szweykowska-Kulinska Z, Beier H
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Würzburg, FRG.
Nucleic Acids Res. 1991 Feb 25;19(4):707-12. doi: 10.1093/nar/19.4.707.
Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.
利用寡核苷酸定向诱变技术，从克隆的拟南芥和烟草tRNA（Tyr）基因中产生琥珀型、赭石型和乳白型抑制基因。无义抑制tRNA（Tyr）基因在HeLa细胞和酵母核提取物中能有效转录，然而，在同源小麦胚提取物以及酵母体外剪接系统中，所有突变前体tRNA（Tyr）的内含子切除都严重受损。抑制性前体tRNA反密码子中一个核苷酸的改变导致潜在的内含子 - 反密码子相互作用发生扭曲。为了证明这导致了剪接效率降低，我们在拟南芥tRNA（Tyr）的内含子中创建了一个点突变，该突变影响了与野生型反密码子的相互作用。正如预期的那样，产生的前体tRNA也剪接效率低下。内含子中的另一个突变恢复了琥珀型反密码子与前体tRNA（Tyr）内含子之间的碱基配对，产生了一种适合小麦胚剪接内切酶的优良底物。这种能产生高水平成熟tRNA（Tyr）的琥珀型抑制tRNA（Tyr）基因，对于研究高等植物中的抑制作用应该是有用的。
