Sherman J M, Söll D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Biochemistry. 1996 Jan 16;35(2):601-7. doi: 10.1021/bi951602b.
Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding. However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2. We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln. Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified. GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln. Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested. A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested. The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo. We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA. On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs.
特定的蛋白质 - 核酸相互作用通常是序列依赖性氢键作用的产物。然而，在大肠杆菌谷氨酰胺 - tRNA合成酶（GlnRS）与tRNAGln复合物的晶体结构中，亮氨酸136（Leu136）通过A72和G2之间的堆积作用稳定了tRNAGln中较弱的第一个（U1 - A72）碱基对的破坏。我们通过体内和体外联合突变分析表明，尽管Leu136与tRNAGln没有形成氢键，但它对tRNA特异性很重要。已经鉴定出GlnRS的更多（L136F）和更少（L136V、L136M、L136A和L136T）错配突变体。GlnRS（L136F）在体内比野生型GlnRS更易发生错配且特异性更低，这不是因为对非同源tRNA的亲和力增加，而是因为对tRNAGln的亲和力降低。此外，与已表征的其他GlnRS错配突变体不同，它在体内并不表现出普遍松弛的tRNA特异性，仅对错配所测试的一部分tRNA起作用。提示了对Py1 - Pu72/Pu2 - Py71组合可能的序列偏好。L136A/M/T/V突变体是在pBR322上表达的首批GlnRS变体，包括野生型，它们在体内不再对错配tyrT（UAG）。我们已经表明，虽然L136A突变体在体内和体外比野生型错配更少，但它并没有更高的特异性，因为它对其同源谷氨酰胺tRNA的亲和力也降低了。基于这些结果，我们认为氨酰 - tRNA合成酶已经进化到能够平衡同源tRNA识别和对非同源tRNA的区分。
