Reevaluation of the d-Amino Acid Compatibility with the Elongation Event in Translation

Abstract

The compatibility of D-amino acids with peptide elongation during translation has been examined in several studies. However, some of the studies have reported that D-amino acids are incompatible with translation, whereas others have reported that D-amino acids are incorporated into polypeptides. Here, we have reevaluated the incorporation of a series of D-amino acids into the nascent chain of short peptides with a reprogrammed genetic code by using the flexible in vitro translation (FIT) system. The FIT system enables the compatibility of each D-amino acid with elongation to be assessed quantitatively in the absence of potential competitors. The incorporation efficiencies were determined by Tricine-SDS-PAGE and the full-length peptide was detected by MALDI-TOF-MS. The D-amino acids were categorized into three groups based on their incorporation efficiencies relative to the corresponding L-amino acid. The D-isomers in group I showed efficiencies of 40% or higher (Ala, Ser, Cys, Met, Thr, His, Phe, and Tyr), and those in group II showed efficiencies of 10-40% (Asn, Gln, Val, and Leu). The D-amino acids in group III produced truncated peptides or no detectable full-length peptides (Arg, Lys, Asp, Glu, Ile, Trp, and Pro). When group I D-amino acids were used consecutively or were alternated with L-amino acids, this completely inhibited their elongation. However, when two or three L-amino acids were inserted between the D-amino acids, the double-incorporation efficiency was restored. Our results quantitatively reveal the compatibility of D-amino acids with peptide elongation and raise new questions about the mechanism of D-amino acid selection and incorporation by the ribosome.

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

“…1 A and B and Table S1). Consistent with previous reports that D-Phe is a substrate for the TM (14), D-Phe-tRNA Phe reached a nearly identical endpoint as L-Phe-tRNA Phe in an EF Tu catalyzed ( Fig. S2 B and C) dipeptide synthesis reaction, albeit one with an apparent rate of synthesis of fMet-D-Phe dipeptide k app of 0.020 s −1 ( Fig.…”

“…During continuous translation, however, P site-bound peptidyl-D-aa-tRNAs partition ECs into two subpopulations, a productive subpopulation that is competent for further rounds of translation elongation and a nonproductive subpopulation that is translationally arrested. This arrested subpopulation, which results in truncated polypeptide products containing a D-amino acid at their C termini due to the failure of the P site-bound peptidyl-D-aa-tRNA to act as a peptidyl-transferase donor, provides the most likely explanation for the poor unnatural amino acid incorporation efficiencies that have been observed during attempts to synthesize proteins containing D-Tyr (8), D-Trp (14), D-Arg (14), and possibly other unnatural amino acids (5).…”

“…Surprisingly, but perhaps explaining early reports of the poor incorporation efficiency of D-Tyr on natural tRNA Tyr (8,13) and more recent reports of the poor incorporation efficiency of D-Phe on an engineered tRNA (14), synthesis of fMet-D-Phe-Lys tripeptide does not go to completion. Instead the reaction plateaus with an apparent endpoint of only 18% fMet converted to fMet-D-Phe-Lys tripeptide with a concomitant accumulation of fMet-D-Phe dipeptide and an apparent rate of 0.004 s −1 ( Fig.…”

“…Biochemical studies with these same aa-tRNAs demonstrated that D-Tyr-tRNA Tyr bound more weakly to elongation factor (EF) Tu than L-Tyr-tRNA Tyr and that D-Tyr was incorporated into the nascent polypeptide by the ribosome at a slower rate and with a lower yield than L-Tyr (13). More recent studies have used engineered suppressor tRNAs (i.e., in vitro transcribed tRNAs lacking all posttranscriptional modifications that read through stop or sense codons) and have reported D-amino acid incorporation efficiencies ranging from 0% to greater than 40% (4,5,14,15), depending on the in vitro translation system used (i.e., comprised of a cell extract or of fully purified components), the identities of the engineered tRNA and D-amino acid used, and the inclusion or exclusion of DTD.…”

“…Motivated by the observation that the TM can incorporate D-amino acids into proteins but does so with relatively low incorporation efficiencies (4,5,14,15), we sought to elucidate at which step(s) during the elongation cycle D-amino acids are discriminated against by the TM and determine the mechanism through which this discrimination occurs. To this end, we have prepared D-aa-tRNAs and evaluated their performance as substrates for the TM during discrete steps of the elongation cycle using a highly active in vitro translation system composed of purified translation components and lacking DTD (16).…”