The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

Abstract

The cellular translational machinery (TM) synthesizes proteins using exclusively L-or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aatRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA 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, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.A lthough the ribosome catalyzes protein synthesis using more than 20 chemically diverse natural amino acids, these natural amino acids are all of L-or achiral configurations. As a consequence, the ability of the ribosome to incorporate L-aminoacyltRNAs (L-aa-tRNAs) with both a high degree of speed and accuracy has been the focus of decades of intense mechanistic and structural investigations (1-3). In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8...

Exploring the Minimal RNA Substrate of Flexizymes

“…This structural information led us to hypothesize that the tRNA helical portion might not be required for flexizyme‐mediated acylation with tRNA analogues. To confirm the veracity of this hypothesis, we compared MhRNA and 4bRNA (5′‐GCCA‐3′) as substrates of flexizymes (dFx and eFx; Figure A) because they were most commonly used for tRNA acylation in previously published studies –…”

“…Conversely, flexizyme tolerance toward amino acids is based on simple recognition of the aromatic groups in the substrates. The flexizyme eFx, optimized for weakly activated l ‐phenylalanine ( l ‐phenylalanine cyanomethyl ester, l ‐Phe‐CME), can catalyze the acylation of tRNA with various aromatic amino acids, including those with different side chains (e.g., l ‐Phe, l ‐Trp, and l ‐Tyr), with the d configuration (e.g., d ‐Phe, d ‐Trp, and d ‐Tyr), and with N‐modified backbones (e.g., N ‐methyl‐Phe, N ‐acyl‐Phe, and N ‐phenyl‐Gly) . Because the recognition elements are present only on the aromatic groups, the CME moiety in an amino acid substrate was further replaced with a p ‐chlorobenzyl thioester (CBT) to transfer the aromatic recognition moiety from the side chain to the leaving group.…”

“…Further in vitro flexizyme selection resulted in dFx, optimized for an amino acid substrate with a DBE moiety. Since then, dFx has been used to synthesize acyl‐tRNAs with amino acids bearing nonaromatic side chains, including those with different side chains (e.g., l ‐Ala, l ‐Ser, and l ‐Arg), with the d ‐configuration (e.g., d ‐Ala, d ‐Ser, and d ‐Arg), with N‐modified backbones (e.g., N ‐acyl‐Lys, N ‐methyl‐Ala, and N ‐alkyl‐Gly), and also with methylene‐extended backbones [e.g., l ‐β‐homoIle ( l ‐βhIle), l ‐β‐homoPhg ( l ‐βhPhg), and l ‐β‐homoGlu ( l ‐βhGlu)] …”

Rewiring protein synthesis: From natural to synthetic amino acids

“…Hecht and colleagues tested ribosome variants with mutations in the peptidyl-transferase center, and found several ribosome mutants with substantially increased efficiency to incorporate D- amino acids into a reporter protein in vitro [77]. A recent study by Englander et al provided mechanistic insights into discrimination of D- amino acids by the wild-type ribosome [78]. It was shown that D -aa-tRNAs are accepted by the ribosomal A site and used as substrates for peptide formation.…”

“…It was shown that D -aa-tRNAs are accepted by the ribosomal A site and used as substrates for peptide formation. However, ribosomes with D -amino acid in the peptidyl-tRNA (P) site are partitioned into translating and arrested subpopulations, therefore impeding further peptide elongation [78]. …”