Bare L A, Uhlenbeck O C
Biochemistry. 1986 Sep 23;25(19):5825-30. doi: 10.1021/bi00367a072.
The aminoacylation kinetics of 19 different variants of yeast tRNATyr with nucleotide substitutions in positions 33-35 were determined. Substitution of the conserved uridine-33 does not alter the rate of aminoacylation. However, substitution of the anticodon position 34 or position 35 reduces Km from 2- to 10-fold and Vmax as much as 2-fold, depending on the nucleotide inserted. The ochre and amber suppressor tRNAsTyr both showed about a 7-fold reduction in Vmax/Km. Data from tRNATyr with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of psi-35. The effect of simultaneous substitutions at positions 34 and 35 can be predicted reasonably well by combining the effects of single substitutions. These data suggest that yeast tyrosyl-tRNA synthetase interacts with positions 34 and 35 of the anticodon of tRNATyr and opens the possibility that nonsense suppressor efficiency may be mediated by the level of aminoacylation.
测定了酵母tRNATyr在33 - 35位核苷酸发生替换的19种不同变体的氨酰化动力学。保守的尿苷-33被替换不会改变氨酰化速率。然而，反密码子34位或35位的替换会使Km降低2至10倍，Vmax降低多达2倍，这取决于插入的核苷酸。赭石型和琥珀型抑制tRNAsTyr的Vmax/Km均降低了约7倍。来自35位具有不同修饰核苷酸的tRNATyr的数据表明，合成酶与假尿苷-35的N1和N3氢之间形成了特定的氢键。通过结合单替换的效应，可以较好地预测34位和35位同时替换的效果。这些数据表明，酵母酪氨酰-tRNA合成酶与tRNATyr反密码子的34位和35位相互作用，并开启了无义抑制效率可能由氨酰化水平介导的可能性。
