Pütz J, Puglisi J D, Florentz C, Giegé R
Laboratoire de Biochimie, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Science. 1991 Jun 21;252(5013):1696-9. doi: 10.1126/science.2047878.
The nucleotides crucial for the specific aminoacylation of yeast tRNA(Asp) by its cognate synthetase have been identified. Steady-state aminoacylation kinetics of unmodified tRNA transcripts indicate that G34, U35, C36, and G73 are important determinants of tRNA(Asp) identity. Mutations at these positions result in a large decrease (19- to 530-fold) of the kinetic specificity constant (ratio of the catalytic rate constant kcat and the Michaelis constant Km) for aspartylation relative to wild-type tRNA(Asp). Mutation to G10-C25 within the D-stem reduced kcat/Km eightfold. This fifth mutation probably indirectly affects the presentation of the highly conserved G10 nucleotide to the synthetase. A yeast tRNA(Phe) was converted into an efficient substrate for aspartyl-tRNA synthetase through introduction of the five identity elements. The identity nucleotides are located in regions of tight interaction between tRNA and synthetase as shown in the crystal structure of the complex and suggest sites of base-specific contacts.
已鉴定出对酵母天冬氨酸tRNA（tRNA(Asp)）由其同源合成酶进行特异性氨酰化至关重要的核苷酸。未修饰的tRNA转录本的稳态氨酰化动力学表明，G34、U35、C36和G73是tRNA(Asp)身份的重要决定因素。这些位置的突变导致相对于野生型tRNA(Asp)，天冬氨酸化的动力学特异性常数（催化速率常数kcat与米氏常数Km之比）大幅降低（19至530倍）。D茎内突变为G10 - C25使kcat/Km降低了八倍。这第五个突变可能间接影响高度保守的G10核苷酸向合成酶的呈现。通过引入五个身份元件，将酵母苯丙氨酸tRNA（tRNA(Phe)）转化为天冬氨酰 - tRNA合成酶的有效底物。如复合物的晶体结构所示，身份核苷酸位于tRNA与合成酶紧密相互作用的区域，并提示了碱基特异性接触的位点。
