Ribosomal incorporation of unnatural amino acids: lessons and improvements from fast kinetics studies

Technologies for genetically programming ribosomal incorporation of unnatural amino acids are expanding and have created many exciting applications. However, these applications are generally limited by low efficiencies of the unnatural incorporations. Here we review our current mechanistic understanding of these limitations delineated from in vitro fast kinetics. Rate limitation occurs by different mechanisms, depending on the classes of the unnatural amino acids and the tRNA adaptors. This new understanding has led to several ways of improving the incorporation efficiencies, as well as challenges of dogma on rate-limiting steps in protein synthesis in natural cells.