Weygand-Durasević I, Rogers M J, Söll D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114.
J Mol Biol. 1994 Jul 8;240(2):111-8. doi: 10.1006/jmbi.1994.1425.
Escherichia coli glutaminyl-tRNA synthetase (GlnRS) specifically recognizes nucleotides in the anticodon and acceptor stem of tRNA(Gln). Extensive conformational changes in the tRNA(Gln):GlnRS complex and requirement for tRNA in glutaminyl-adenylate formation suggests that accurate anticodon recognition is required for aminoacylation. A 17 amino acid loop in GlnRS (residues 476 to 492) that connects two beta-ribbon motifs was targeted for saturation mutagenesis as the motifs span the anticodon binding domain and extend to the active site. Opal suppressor tRNAs (GLN) derived from tRNA(Gln) are poor substrates for GlnRS, and compensating mutations in glnS (the structural gene for GlnRS) were selected by the ability of the mutant gene product to aminoacylate such a suppressor (GLNA3U70). A number of mutations in loop 476 to 492 were identified by genetic selection, and two of the GlnRS purified mutant enzymes showed elevated specificity constants (kcat/Km) for aminoacylation of a tRNA(Gln)-derived transcript with the opal (UCA) anticodon when compared with the wild-type enzyme. The specificity constants for the mutant enzymes with the cognate tRNA(Gln) transcript (anticodon CUG) were unchanged. Therefore, region 476 to 492 has been identified in communicating anticodon recognition with the active site at a distance of more than 30 A away, supporting a proposed model from the structure of the complex between tRNA(Gln):GlnRS. A previous study has identified residues that interact with the inside of the L-shaped tRNA as communicating accurate anticodon recognition. Therefore, at least two pathways of communication have been identified in the accurate recognition of tRNA by GlnRS.
大肠杆菌谷氨酰胺-tRNA合成酶（GlnRS）特异性识别tRNA(Gln)反密码子和受体茎中的核苷酸。tRNA(Gln):GlnRS复合物中广泛的构象变化以及谷氨酰胺-腺苷酸形成过程中对tRNA的需求表明，精确的反密码子识别是氨酰化所必需的。GlnRS中一个连接两个β- ribbon基序的17个氨基酸的环（第476至492位残基）被作为饱和诱变的靶点，因为这些基序跨越反密码子结合结构域并延伸至活性位点。源自tRNA(Gln)的乳白抑制tRNA（GLN）是GlnRS的劣质底物，通过突变基因产物对这种抑制tRNA（GLNA3U70）进行氨酰化的能力来选择glnS（GlnRS的结构基因）中的补偿性突变。通过遗传筛选鉴定出了第476至492位环中的一些突变，与野生型酶相比，两种纯化的GlnRS突变酶对具有乳白（UCA）反密码子的tRNA(Gln)衍生转录本进行氨酰化时，其特异性常数（kcat/Km）升高。突变酶对同源tRNA(Gln)转录本（反密码子CUG）的特异性常数没有变化。因此，已确定第476至492位区域在距离超过30 Å的位置将反密码子识别与活性位点进行沟通，这支持了基于tRNA(Gln):GlnRS复合物结构提出的模型。先前的一项研究已鉴定出与L形tRNA内部相互作用的残基，这些残基在传递精确的反密码子识别信息。因此，在GlnRS对tRNA的精确识别中已鉴定出至少两条沟通途径。
