Plant nonsense suppressor tRNATyr genes are expressed at very low leves in vitro due to inefficient splicing of the intron-containing pre-tRNAS

摘要

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabldopsk and Nicotiana tRNATyr genes. The nonsense suppressor tRNATyr genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAsTyr was severely Impaired in the homologous wheat germ extract as well as in the yeast in vitro spliclng system. The change of one nucieotide 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 Arabldopsis tRNATyr 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 pretRNATyr, resulted in an excellent substrate for wheat germ splicing endonuciease. This type of amber suppressor tRNATyr gene which yields high levels of mature tRNATyr should be useful for studying suppression in higher plants.