Fender Aurélie, Sissler Marie, Florentz Catherine, Giegé Richard
Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René-Descartes, 67084 Strasbourg cedex, France.
Biochimie. 2004 Jan;86(1):21-9. doi: 10.1016/j.biochi.2003.11.011.
The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.
同源氨酰 - tRNA合成酶对转运RNA（tRNA）进行氨酰化的特异性是合成功能性蛋白质的关键步骤。已确定单个tRNA或一组同功tRNA的氨酰化特性与允许同源合成酶识别的正信号（决定因素）以及导致非同源合成酶排斥的负信号（反决定因素）的存在有关。通常通过将相应的核苷酸移植到一个或几个宿主tRNA中来测试身份集的完整性，移植后的宿主tRNA会因此获得新的氨酰化特异性。此类移植实验对于检测宿主tRNA中给定氨酰化身份集的最佳表达所需的特殊结构优化也很有用。本研究探索了将已定义的酵母天冬氨酸身份集表达于具有相同特异性的不同tRNA支架中，即四种酵母tRNA（Arg）同功受体。目的是研究由于宿主tRNA的独特特异性，新身份的表达是否相似，或者由于它们独特的序列和结构特征，其表达是否存在差异。制备了体外转录的天然tRNA（Arg）同功受体以及携带天冬氨酸身份元件的变体，并确定了它们的氨酰化特性。四种野生型同功受体在精氨酰化反应中具有活性，催化效率在20倍范围内，而在天冬氨酰化反应中无活性。虽然移植后的tRNA（1）（Arg）和tRNA（4）（Arg）转变为酵母天冬氨酰 - tRNA合成酶的高效底物，但移植后的tRNA（2）（Arg）和tRNA（3）（Arg）的天冬氨酰化程度仍然很低。在这两种tRNA中寻找反决定因素揭示了其独特特征。将单个碱基对C6 - G67转换为tRNA（Asp）中存在的碱基对G6 - C67，可使移植后的tRNA（2）（Arg）中天冬氨酸身份完全表达，但在tRNA（3）（Arg）中则不然。得出的结论是，不同的同功受体tRNA通过独特的反决定途径保护自身免受错误氨酰化。
