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-aa-tRNA 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.

Keywords: D-amino acids; D-aminoacyl-tRNA deacylase; ribosome; translation arrest.

Fig. 1.

D-Phe-tRNA^Phe exhibits translation disorders during peptide elongation. (A) Synthesis of dipeptide f-[³⁵S]-Met-L-Phe (Left) and f-[³⁵S]-Met-D-Phe (Right) versus time. Reaction products were separated using eTLC. (B) Plot of the fraction of f-[³⁵S]-Met converted to f-[³⁵S]-Met-X versus time where X = L-Phe (black squares) or D-Phe (gold squares) from A. Experiments were run in duplicate, and the SE between the two measurements is reported. (C) Synthesis of tripeptide f-[³⁵S]-Met-L-Phe-Lys (Left) and f-[³⁵S]-Met-D-Phe-Lys (Right) versus time. Reaction products were separated using eTLC. (D) Plot of the fraction of f-[³⁵S]-Met converted to f-[³⁵S]-Met-X-Lys versus time where X = L-Phe (black squares) or D-Phe (gold squares) from C. Experiments were run in duplicate, and the SE between the two measurements is reported. (E) Synthesis of f-[³⁵S]-Met-D-Phe-Lys tripeptide and f-[³⁵S]-Met-D-Phe-Lys-Glu tetrapeptide. Translation reactions were allowed to proceed for 30 min, and reaction products were separated using eTLC.

Fig. 2.

D-Phe-tRNA^Phe inhibits protein synthesis in the ribosomal PTC. (A) Nitrocellulose filter binding assays. The fraction of dipeptide f-[³⁵S]-Met-L-Phe-tRNA^Phe (black squares) and f-[³⁵S]-Met-D-Phe-tRNA^Phe (gold squares) bound versus time. Experiments were performed in the presence of EF-G. Experiments were run in triplicate, and the SD between the measurements is reported. (B) Denaturing PAGE (d-PAGE) analysis of toeprinting assays. Lane 1, ribosomal initiation complexes; lane 2, ribosomal initiation complexes that have been incubated with L-Phe-tRNA^Phe ternary complexes; lanes 3 and 4, ribosomal initiation complexes that have been incubated with L-Phe-tRNA^Phe ternary complexes and EF-G for 15 s (lane 3) and 20 min (lane 4); lanes 5–10, ribosomal initiation complexes that have been incubated with D-Phe-tRNA^Phe ternary complexes and EF-G for 15 s (lane 5) up through 20 min (lane 10). (C) Plot of the fraction of f-Met-L-Phe-tRNA^Phe (black squares) and f-Met-D-Phe-tRNA^Phe (gold squares) translocated versus time from B. Experiments were run in duplicate, and the SE is reported. (D) Pmn reactivity of f-[³⁵S]-Met-L-Phe-tRNA^Phe (Left) and f-[³⁵S]-Met-D-Phe-tRNA^Phe (Right) versus time. Reaction products were separated using eTLC. (E) Plot of the fraction of f-[³⁵S]-Met converted to to f-[³⁵S]-Met-X–Pmn where X = L-Phe (black squares) or D-Phe (gold squares) versus time from D. Experiments were run in duplicate, and the SE is reported.

Fig. 3.

The presence of a D-amino acid at the C terminus of a P site-bound peptidyl-tRNA alters the conformation of the ribosome. (A) Chemical protection assays for ECs carrying either fMet-L-Phe-tRNA^Phe or fMet-D-Phe-tRNA^Phe. Lane 1, unmodified ECs carrying P site-bound fMet-L-Phe-tRNA^Phe; lane 2, DMS-modified vacant ribosomes (i.e., not carrying any tRNAs); lane 3, DMS-modified ECs carrying P site-bound fMet-L-Phe-tRNA^Phe; lane 4, DMS-modified ECs carrying P site-bound fMet-D-Phe-tRNA^Phe. (B) Average structures of the PTCs of ECs carrying either P site-bound fMet-L-Phe-tRNA^Phe and A site-bound Lys-tRNA^Lys (shown in black) or P site-bound fMet-D-Phe-tRNA^Phe and A site-bound Lys-tRNA^Lys (shown in gold) were calculated based on the last 30-ns trajectories of MD simulations. PTC nucleotides are shown in light purple (P site-bound fMet-L-Phe-tRNA^Phe) and dark purple (P site-bound fMet-D-Phe-tRNA^Phe).

Fig. 4.

Mechanistic scheme for the partitioning of ECs carrying a P site-bound peptidyl-D-aa-tRNA into translationally arrested and translationally competent subpopulations. Delivery and incorporation of D-aa-tRNA result in the effective formation of an EC carrying a P site-bound peptidyl-D-aa-tRNA. During the next round of continuous translation, the presence of a P site-bound peptidyl-D-aa-tRNA partitions ECs into two subpopulations, a productive subpopulation that is competent for further rounds of translation elongation and a nonproductive subpopulation that is translationally arrested.