Event

The Mechanisms Underlying the Substrate Specificity of the Translation Machinery

Abstract

The translation machinery (TM) is unique in nature in that it has evolved to use twenty chemically distinct substrates in a nearly equivalent manner, thus enabling it to use different linear combinations of the twenty natural amino acids to synthesize the great diversity of proteins that are found in all living cells. Inspired by the inherent ability of the TM to use a relatively broad range of substrates, synthetic biologists have sought to further expand the substrate specificity of the TM to include unnatural amino acids. These efforts hold great promise for: generating uniquely modified proteins that can be used to investigate biological mechanisms; evolving ‘designer’ proteins that expand the repertoire of chemical reactivities available to conventional proteins; designing and developing innovative, peptide-based therapeutics; and synthesizing entirely novel polymers for applications in industries ranging from pharmaceuticals to nanotechnology. Despite this great promise and several key advances in this area of synthetic biology, many useful unnatural amino acids can apparently not be incorporated by the TM and the mechanistic basis for the discrimination and rejection of these substrates by the TM remains unknown. Using a combination of chemical biology, biochemistry, single-molecule fluorescence microscopy, and molecular dynamics simulations, we have developed a framework for investigating the substrate specificity of the TM and have used this framework to determine the mechanistic basis for the impaired incorporation efficiency of D-amino acids, a paradigmatic class of unnatural amino acids. Our results reveal a novel mechanism through which D-aminoacyl-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aminoacyl-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aminoacyl-tRNAs from cells.