Outwitting EF-Tu and the ribosome: Translation with D-amino acids

Abstract

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of D-amino acids (D-aa) into polypeptides. The rare appearance of D-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitrotranslation system that enables single incorporation of 17 out of 18 tested D-aa into a polypeptide; incorporation of two or three successive D-aa was also observed in several cases. The system consists of wild-type components and D-aa are introduced via artificially charged, unmodified tRNAGly that was selected according to the rules of 'thermodynamic compensation'. The results reveal an unexpected plasticity of the ribosomal peptidyl-transferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of D-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.