The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center Michael T. Englandera,1, Joshua L. Avinsb,2,3, Rachel C. Fleisherb,3, Bo Liuc, Philip R. Effraima,4, Jiangning Wangb,5, Klaus Schultenc,d,e, Thomas S. Leyhf, Ruben L. Gonzalez Jr.b,6, and Virginia W. Cornishb,6 aIntegrated Program in Cellular, Molecular and Biomedical Sciences, Department of Chemistry, and bDepartment of Chemistry, Columbia University, New York, NY 10027; cCenter for Biophysics and Computational Biology, dDepartment of Physics, and eBeckman Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and fDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461 Edited by Ignacio Tinoco, Jr., University of California, Berkeley, CA, and approved March 24, 2015 (received for review January 8, 2015) The cellular translational machinery (TM) synthesizes proteins using The high fidelity of protein synthesis has historically been at- exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the tributed to the cumulative accuracy with which aaRSs amino- presence of D-amino acids in nature and their ability to be amino- acylate amino acids onto tRNAs (11) and with which the acylated onto tRNAs by aa-tRNA synthetases. The ubiquity of ribosome repetitively selects and incorporates aa-tRNAs in the L-amino acids in proteins has led to the hypothesis that D-amino order dictated by the mRNA (1–3). Once an aa-tRNA has been acids are not substrates for the TM. Supporting this view, protein incorporated into the ribosomal aa-tRNA binding (A) site, the engineering efforts to incorporate D-amino acids into proteins using ribosome positions the carbonyl carbon of the ester bond linking the TM have thus far been unsuccessful. Nonetheless, a mechanistic the nascent polypeptide to the peptidyl-tRNA bound at the ri- understanding of why D-aa-tRNAs are poor substrates for the TM is bosomal peptidyl-tRNA binding (P) site for nucleophilic attack lacking. To address this deficiency, we have systematically tested by the α-amino group of the amino acid moiety of the newly the translation activity of D-aa-tRNAs using a series of biochemical incorporated A-site aa-tRNA and catalyzes the peptidyl-transfer assays. We find that the TM can effectively, albeit slowly, accept reaction (12). Following peptidyl transfer, the newly deacylated D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site P-site tRNA and the newly formed A-site peptidyl-tRNA car- D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the rying the nascent polypeptide that has been extended by one resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA amino acid are translocated together with the mRNA into the binding (P) site. During the next round of continuous translation, ribosomal tRNA exit (E) site and the P site, respectively, pre- however, we find that ribosomes carrying a P-site peptidyl-D-aa- paring the ribosomal complex for the next round of the trans- tRNA partition into subpopulations that are either translationally lation elongation cycle. It is within this framework that the speed arrested or that can continue translating. Consistent with its ability and accuracy with which the TM incorporates L-amino acids into to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA Significance can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, Utilization of the translation machinery (TM) to incorporate un- provide insight into how the TM might be engineered to use natural amino acids (UAAs) to create polymers with novel func- D-aa-tRNAs, and increase our understanding of the physiological tion holds great promise for synthetic biology. Many useful role of a widely distributed enzyme that clears D-aa-tRNAs from cells. UAAs, however, are not substrates for the TM, yet the mecha- nistic bases for the TM’s ability to discriminate against UAAs re- ribosome | D-amino acids | translation arrest | D-aminoacyl-tRNA deacylase main unknown. Here we elucidate the mechanistic basis for the impaired incorporation efficiency of D-amino acids. We show that the ribosome discriminates the chirality of the amino acid within lthough the ribosome catalyzes protein synthesis using more its peptidyl-transferase center (PTC). Our results demonstrate Athan 20 chemically diverse natural amino acids, these natural a novel mechanism through which D-aminoacyl-tRNAs (D-aa- amino acids are all of L- or achiral configurations. As a conse- tRNAs) interfere with translation, provide insight into how the quence, the ability of the ribosome to incorporate L-aminoacyl- TM might be engineered to use D-aa-tRNAs, and suggest addi- tRNAs (L-aa-tRNAs) with both a high degree of speed and ac- tional physiological utility to a widely distributed enzyme that curacy has been the focus of decades of intense mechanistic and clears D-aa-tRNAs from cells. structural investigations (1–3). In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Author contributions: M.T.E., K.S., T.S.L., R.L.G., and V.W.C. designed research; M.T.E., J.L.A., Nonetheless, a comprehensive mechanistic understanding of how R.C.F., and B.L. performed research; J.L.A., R.C.F., P.R.E., and J.W. contributed new reagents/ the translational machinery (TM) responds to D-aa-tRNAs is of analytic tools; M.T.E., J.L.A., R.C.F., P.R.E., B.L., K.S., T.S.L., R.L.G., and V.W.C. analyzed data; and M.T.E., R.C.F., R.L.G., and V.W.C. wrote the paper. interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering The authors declare no conflict of interest. applications that seek to use the synthetic power of the ribosome This article is a PNAS Direct Submission. 1 to create novel polymers (4) as well as for mechanistic applications Present address: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104. that seek to probe protein structure and folding with unnatural 2Present address: Department of Chemistry, University of Illinois at Urbana–Champaign, amino acids (5). Second, there is growing evidence that ribosomes Urbana, IL 61801. may have to contend with D-aa-tRNAs in vivo: D-amino acids are 3J.L.A. and R.C.F. contributed equally to this work. synthesized by racemase enzymes (6) and can be found at high 4Present address: Department of Anesthesiology, Yale University, New Haven, CT 06520. concentrations in cells (7), and a growing number of aa-tRNA 5Present address: MSCF Program, Tepper School of Business, Carnegie Mellon University, synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs New York, NY 10004. with D-amino acids (8, 9). Consistent with this possibility, the 6 To whom correspondence may be addressed. Email: [email protected] or vc114@ D-aa-tRNA deacylase (DTD) enzyme is nearly universally con- columbia.edu. served (10) and functions to remove D-amino acids that have been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. misacylated onto tRNA (8, 10). 1073/pnas.1424712112/-/DCSupplemental. 6038–6043 | PNAS | May 12, 2015 | vol. 112 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1424712112 proteins has been extensively investigated (1–3). Although the ribosome that stabilizes the peptidyl-transferase center (PTC) in an ability of the TM to incorporate D-amino acids into proteins has inactive conformation. been similarly, albeit less extensively, investigated in various in vitro translation systems, these studies have found that D-amino Results acids are incorporated with zero or low incorporation efficiencies. D-aa-tRNAs Are Incorporated by the TM But Can Arrest Actively Early studies using a cell-free, extract-based translation system Translating Ribosomes. We investigated the ability of D-aa-tRNAs devoid of DTD showed that D-Tyr-tRNATyr that had been to be incorporated into short peptides by the TM in a highly pu- prepared using a natural tRNATyr (i.e., a wild-type tRNATyr that rified, highly active in vitro translation system (16) devoid of DTD. had been purified from cells and that presumably contained First, the yields and rates of synthesis of fMet-D-Phe and fMet- all of its naturally occurring posttranscriptional modifications) L-Phe dipeptides were compared by measuring the percent of f-[35S]- could be incorporated by the TM, but with only an ∼17% effi- Met-tRNAfMet converted to peptide product versus time using ciency relative to L-Tyr-tRNATyr prepared with the same natural electrophoretic TLC (eTLC) (17). In these experiments, D- or tRNATyr (8). Biochemical studies with these same aa-tRNAs L-Phe cyanomethyl ester was charged onto natural, posttranscrip- demonstrated that D-Tyr-tRNATyr bound more weakly to elon- tionally modified tRNAPhe using the Flexizyme ribozyme (18) (Figs. gation factor (EF) Tu than L-Tyr-tRNATyr and that D-Tyr was S1 and S2A). Ternary complexes of EF Tu, GTP, and aa-tRNAs incorporated into the nascent polypeptide by the ribosome at a [EF-Tu(GTP)aa-tRNAs] were delivered to limiting quantities of slower rate and with a lower yield than L-Tyr (13). More recent ribosomal initiation complexes. Initiation complexes were prepared studies have used engineered suppressor tRNAs (i.e., in vitro by assembling ribosomes bearing f-[35S]-Met-tRNAfMet at the ri- transcribed tRNAs lacking all posttranscriptional modifications bosomal P site on a variant of the mRNA encoding bacteriophage that read through stop or sense codons) and have reported T4 gene product 32 that commences with fMet-Phe-Lys as the first D-amino acid incorporation efficiencies ranging from 0% to 3 of the 20 amino acids that this variant mRNA encodes.