Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California at Santa Cruz, Santa Cruz, CA 95064, USA.
Structure. 2013 Jul 2;21(7):1258-63. doi: 10.1016/j.str.2013.04.028. Epub 2013 Jun 13.
Bacterial translation termination is mediated by release factors RF1 and RF2, which recognize stop codons and catalyze hydrolysis of the peptidyl-tRNA ester bond. The catalytic mechanism has been debated. We proposed that the backbone amide NH group, rather than the side chain, of the glutamine of the universally conserved GGQ motif participates in catalysis by H-bonding to the tetrahedral transition-state intermediate and by product stabilization. This was supported by complete loss of RF1 catalytic activity when glutamine is replaced by proline, the only residue that lacks a backbone NH group. Here, we present the 3.4 Å crystal structure of the ribosome complex containing the RF2 Q253P mutant and find that its fold, including the GGP sequence, is virtually identical to that of wild-type RF2. This rules out proline-induced misfolding and further supports the proposal that catalytic activity requires interaction of the Gln-253 backbone amide with the 3' end of peptidyl-tRNA.
细菌翻译终止是由释放因子 RF1 和 RF2 介导的，它们识别终止密码子并催化肽酰-tRNA 酯键的水解。催化机制一直存在争议。我们提出，普遍保守的 GGQ 基序中谷氨酰胺的酰胺 NH 基团而不是侧链参与催化，通过氢键与四面体型过渡态中间体和产物稳定化相互作用。这一假设得到了充分的支持，因为当谷氨酰胺被脯氨酸取代时，RF1 的催化活性完全丧失，脯氨酸是唯一缺乏酰胺 NH 基团的残基。在这里，我们展示了含有 RF2 Q253P 突变体的核糖体复合物的 3.4Å 晶体结构，发现其折叠结构，包括 GGP 序列，与野生型 RF2 几乎完全相同。这排除了脯氨酸诱导的错误折叠，并进一步支持了这样的假设，即催化活性需要 Gln-253 酰胺的酰胺与肽酰-tRNA 的 3' 末端相互作用。
