Thioesters Support Efficient Protein Biosynthesis by the Ribosome

Abstract

Thioesters are critical chemical intermediates in numerous extant biochemical reactions and are invoked as key reagents during prebiotic peptide synthesis on an evolving Earth. Here we asked if a thioester could replace the native oxo-ester in acyl-tRNA substrates during protein biosynthesis by the ribosome. We prepared 3′-thio-3′-deoxyadenosine triphosphate in 10 steps from xylose and demonstrated that it is an effective substrate for the Escherichia coli CCA-adding enzyme, which appends 3′-thio-3′-deoxyadenosine to truncated tRNAs ending with 3′-CC. Using a variety of aminoacyl-tRNA synthetases, flexizymes, or a direct thioester exchange reaction, we prepared a suite of 3′-thio-tRNAs acylated with α- and non-α-amino acids. All were recognized and utilized by wild-type E. coli ribosomes during in vitro translation reactions to generate oligopeptides in yields commensurate with native oxo-ester tRNAs. These results indicate that thioester intermediates widely used in Nature can be co-opted to support the incorporation of natural α-amino acids as well as noncanonical monomers by the extant translational machinery for sequence-defined polymer synthesis.

We show that tRNAs acylated with a thioester rather than an ester linkage to natural and unnatural monomers can be used by the extant translation machinery for sequence-defined polymer synthesis.