Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro

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• 作者：Jinfan Wang ; Marek Kwiatkowski ; Michael Y. Pavlov ; M氓ns Ehrenberg ; Anthony C. Forster
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：June 20, 2014
• 年：2014
• 卷：9
• 期：6
• 页码：1303-1311
• 全文大小：435K
• 年卷期：v.9,no.6(June 20, 2014)
• ISSN：1554-8937

文摘

Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA^Pro, increases translation incorporation rates of all three studied NMAAs compared with tRNA^Phe- and tRNA^Ala-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.