Initiating translation with <i>D</i> -amino acids

<jats:p> Here we report experimental evidence that the translation initiation apparatus accepts <jats:italic>D</jats:italic> -amino acids ( <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa), as opposed to only <jats:italic>L</jats:italic> -methionine, as initiators. Nineteen <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa, as the stereoisomers to their natural <jats:italic>L</jats:italic> -amino acids, were charged onto initiator tRNA <jats:sup>fMet</jats:sup> <jats:sub>CAU</jats:sub> using flexizyme technology and tested for initiation in a reconstituted <jats:italic>Escherichia coli</jats:italic> translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa. Remarkably, all <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa-tRNA <jats:sup>fMet</jats:sup> <jats:sub>CAU</jats:sub> . Although the inefficient formylation of <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa-tRNA <jats:sup>fMet</jats:sup> <jats:sub>CAU</jats:sub> resulted in modest expression of the corresponding peptide, preacetylation of <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa-tRNA <jats:sup>fMet</jats:sup> <jats:sub>CAU</jats:sub> dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa. Our findings provide not only the experimental evidence that translation initiation tolerates <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa, but also a new means for the mRNA-directed synthesis of peptides capped with <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa or acyl- <jats:italic> <jats:sup>D</jats:sup> </jats:italic> aa at the N terminus. </jats:p>