Bedouelle H, Nageotte R
Groupe d'Ingénierie des Protéines (CNRS URA 1129), Institut Pasteur, Paris, France.
EMBO J. 1995 Jun 15;14(12):2945-50. doi: 10.1002/j.1460-2075.1995.tb07294.x.
In the process of genetic translation, each aminoacyl-tRNA synthetase specifically aminoacylates its cognate tRNAs and rejects the 19 other species of tRNAs. A decrease in the specificity of this reaction can result in misincorporations of amino acids into proteins and be deleterious to the cell. In the case of tyrosyl-tRNA synthetase from Bacillus stearothermophilus, the change of residue Glu152 into Ala results in erroneous interactions with non-cognate tRNAs. To analyse how Glu152 contributes to the discrimination between tRNAs by tyrosyl-tRNA synthetase, 11 changes to this residue were created by mutagenesis. The misaminoacylations of tRNA(Phe) and tRNA(Val) with tyrosine in vitro (on a scale going from 1 to 30) and the toxicity of tyrosyl-tRNA synthetase in vivo (on a scale from 1 to 10(7)) increased in a correlated way when the nature of the side chain in position 152 varied from negatively charged to uncharged then to positively charged. The aminoacylation of tRNA(Tyr) was unaffected by the mutations. The results show that the role of Glu152 in the discrimination between tRNAs is purely negative, that it acts by electrostatic repulsion of non-cognate tRNAs and that this mechanism has been conserved throughout evolution.
在基因翻译过程中，每种氨酰 - tRNA合成酶都能特异性地将其对应的tRNA氨酰化，并排斥其他19种tRNA。该反应特异性的降低会导致氨基酸错误掺入蛋白质中，对细胞有害。就嗜热栖热芽孢杆菌的酪氨酰 - tRNA合成酶而言，将第152位残基谷氨酸替换为丙氨酸会导致与非对应tRNA发生错误相互作用。为了分析第152位谷氨酸残基如何有助于酪氨酰 - tRNA合成酶区分不同的tRNA，通过诱变对该残基进行了11种替换。当第152位侧链的性质从带负电荷变为不带电荷再变为带正电荷时，体外tRNA（Phe）和tRNA（Val）被酪氨酸错误氨酰化的程度（范围为1至30）以及体内酪氨酰 - tRNA合成酶的毒性（范围为1至10⁷）以相关方式增加。tRNA（Tyr）的氨酰化不受这些突变的影响。结果表明，第152位谷氨酸残基在区分tRNA中的作用纯粹是负面的，它通过对非对应tRNA的静电排斥起作用，并且这种机制在整个进化过程中一直保留。
