Himeno H, Hasegawa T, Ueda T, Watanabe K, Shimizu M
Institute of Space and Astronautical Science, Kanagawa, Japan.
Nucleic Acids Res. 1990 Dec 11;18(23):6815-9. doi: 10.1093/nar/18.23.6815.
The discrimination mechanism between tRNA(Ser) and tRNA(Tyr) was studied using various in vitro transcripts of E. coli tRNATyr variants. The insertion of only two nucleotides into the variable stem of tRNA(Tyr) generates serine charging activity. The acceptor activities of some of the tRNA(Tyr) mutants with insertions in the long variable arm were enhanced by changes in nucleotides at positions 9 and/or 20B, which are possible elements for dictating the orientation of the long variable arm. These findings suggest that the long variable arm is involved in recognition by seryl-tRNA synthetase in spite of sequence and length variations shown within tRNA(Ser) isoacceptors, and eventually serves as a determinant for selection from other tRNA species. Changing the anticodon from GUA to the serine anticodon GGA resulted in a marked decrease in tyrosine charging activity, but this mutant did not show any serine charging activity. The discriminator base, the fourth base from the 3' end of tRNA, was also important for aminoacylation with tyrosine. Complete specificity change in vitro was facilitated by insertion of three nucleotides into the variable arm plus two nucleotide changes at positions 9 and 73.
利用大肠杆菌tRNATyr变体的各种体外转录本研究了tRNA(Ser)和tRNA(Tyr)之间的识别机制。仅在tRNA(Tyr)的可变茎中插入两个核苷酸就会产生丝氨酸负载活性。一些在长可变臂中插入的tRNA(Tyr)突变体的受体活性通过9位和/或20B位核苷酸的变化而增强，这两个位置可能是决定长可变臂方向的元件。这些发现表明，尽管tRNA(Ser)同工受体中存在序列和长度变化，但长可变臂仍参与丝氨酰-tRNA合成酶的识别，最终作为从其他tRNA种类中进行选择的决定因素。将反密码子从GUA改变为丝氨酸反密码子GGA导致酪氨酸负载活性显著降低，但该突变体未表现出任何丝氨酸负载活性。判别碱基，即tRNA 3'端的第四个碱基，对于酪氨酸的氨酰化也很重要。通过在可变臂中插入三个核苷酸以及在9位和73位进行两个核苷酸的改变，促进了体外完全特异性的改变。
