Specificity of translation for N-alkyl amino acids

We examine the specificity of translation for various primary and secondary amino acid analogues. A synthetase-free, pure, E. coli translation system is used to prevent competing reactions, and three different tRNA adaptor:codon pairs are used to control for tRNA- and codon-specific effects. Surprisingly, N -butyl amino acids fail to incorporate, N -methyl amino acid incorporation efficiencies are dependent on the tRNA adaptor, yet hydroxyPro, Pro, Phe, and Ala incorporate near quantitatively from all adaptors. This suggests that Pro is a privileged N -alkyl amino acid for incorporation by the translation apparatus and establishes that very efficient N -methyl amino acid incorporation is possible if matched with an optimal tRNA adaptor. Results support exploration of Pro analogues and N -methyl amino acids as substrates for engineering ribosomal synthesis of genetically selectable libraries of protease-resistant, N -alkyl peptide ligands.