d-Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA.

A complete understanding of the determinants that restrict d-amino acid incorporation by the ribosome, which is of interest to both basic biologists and the protein engineering community, remains elusive. Previously, we demonstrated that d-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA (peptidyl-d-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the d-amino acid side chain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-d-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNA acceptor contribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of d-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing d-amino acids.