Jahn M, Rogers M J, Söll D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Nature. 1991 Jul 18;352(6332):258-60. doi: 10.1038/352258a0.
The correct attachment of amino acids to their corresponding (cognate) transfer RNA catalysed by aminoacyl-tRNA synthetases is a key factor in ensuring the fidelity of protein biosynthesis. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system to study this highly specific recognition process, also referred to as 'tRNA identity'. Accurate acylation of tRNA depends mainly on two principles: a set of nucleotides in the tRNA molecule (identity elements) responsible for proper discrimination by aminoacyl-tRNA synthetases and competition between different synthetases for tRNAs. Elements of glutamine identity are located in the anticodon and in the acceptor stem region, including the discriminator base. We report here the production of more than 20 tRNA(2Gln) mutants at positions likely to be involved in tRNA discrimination by the enzyme. Unmodified tRNA, containing the wild-type anticodon and U or G at its 5'-terminus, can be aminocylated by GlnRS with similar kinetic parameters to native tRNA(2Gln). By in vitro aminoacylation the mutant tRNAs showed decreases of up to 3 x 10(5)-fold in the specificity constant (kcat/KM)14 with the major contribution of kcat. Despite these large changes, some of these mutant tRNAs are efficient amber suppressors in vivo. Our results show that strong elements for glutamine identity reside in the anticodon region and in positions 2 and 3 of the acceptor stem, and that the contribution of different identity elements to the overall discrimination varies significantly. We discuss our data in the light of the crystal structure of the GlnRS:tRNA(Gln) complex.
氨酰 - tRNA合成酶催化氨基酸与相应（同源）转运RNA的正确连接是确保蛋白质生物合成保真度的关键因素。先前的研究表明，大肠杆菌tRNA(Gln)与谷氨酰胺 - tRNA合成酶（GlnRS）的相互作用为研究这种高度特异性的识别过程提供了一个极佳的系统，该过程也被称为“tRNA身份识别”。tRNA的准确氨酰化主要取决于两个原则：tRNA分子中的一组核苷酸（身份元件）负责被氨酰 - tRNA合成酶正确识别，以及不同合成酶对tRNA的竞争。谷氨酰胺身份元件位于反密码子和受体茎区域，包括判别碱基。我们在此报告了在可能参与该酶对tRNA识别的位置产生了20多种tRNA(2Gln)突变体。含有野生型反密码子且其5'端为U或G的未修饰tRNA，可以被GlnRS氨酰化，其动力学参数与天然tRNA(2Gln)相似。通过体外氨酰化，突变tRNA的特异性常数（kcat/KM）下降了高达3×10^5倍，其中kcat的贡献最大。尽管有这些巨大变化，但其中一些突变tRNA在体内是有效的琥珀抑制子。我们的结果表明，谷氨酰胺身份的强元件存在于反密码子区域以及受体茎的第2和第3位，并且不同身份元件对整体识别的贡献差异显著。我们根据GlnRS:tRNA(Gln)复合物的晶体结构来讨论我们的数据。
