Bedouelle H, Guez-Ivanier V, Nageotte R
Groupe d' Ingénierie des Protéines (CNRS-URA 1129), Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Biochimie. 1993;75(12):1099-108. doi: 10.1016/0300-9084(93)90009-h.
We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNA(Tyr) by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRS and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNA(Tyr) and tRNA(Gln) indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNA(Tyr) confirm the model of the TyrRS:tRNA(Tyr) complex on the following points. TyrRS approaches tRNA(Tyr) on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNA(Tyr). We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glu152 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for folding and function, in particular for the specific recognition and charging of tRNA(Tyr).
我们通过连续的预测、酪氨酰 - tRNA合成酶（TyrRS）的诱变和分子建模构建了嗜热脂肪芽孢杆菌的酪氨酰 - tRNA合成酶（TyrRS）与tRNA（Tyr）之间复合物的模型。我们将这个模型与独立获得的数据进行对比，将其与其他复合物的晶体结构进行比较，并回顾了关于TyrRS对tRNA进行区分的最新数据。TyrRS和GlnRS（两者均为I类合成酶）晶体结构的比较，以及tRNA（Tyr）和tRNA（Gln）的同一性元件的比较表明，这两种合成酶结合其同源tRNA的方式不同。关于tRNA（Tyr）的诱变数据在以下几点上证实了TyrRS:tRNA（Tyr）复合物的模型。TyrRS从可变环一侧接近tRNA（Tyr）。接受茎前三对碱基未被识别。C6位存在NH2基团以及C2位不存在庞大基团对于TyrRS识别判别碱基A73很重要，这仅在酰基转移的过渡态中才完全实现。反密码子是tRNA（Tyr）的主要同一性元件。我们建立了一种体内方法来研究合成酶突变对tRNA区分的影响。使用这种方法，我们已经表明，TyrRS的Glu152残基通过静电和空间排斥作用，对非同源tRNA起到纯粹的负向判别作用。来自大肠杆菌和嗜热脂肪芽孢杆菌的野生型TyrRS的过量表达对大肠杆菌有毒，这是由于tRNA的错误氨基酸负载或非生产性结合。大肠杆菌和嗜热脂肪芽孢杆菌的TyrRS之间一系列杂种的构建表明，它们的序列和结构在进化过程中在局部保持了兼容性，以实现折叠和功能，特别是对于tRNA（Tyr）的特异性识别和氨基酸负载。
