Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation

Abstract

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.

Figure 1.

Schematic illustration of the interaction between EF-Tu and aminoacyl-tRNAs. (A) EF-Tu-mediated delivery of pAA-tRNA into the ribosome. (B) A potential steric clash of the N-methyl group of ^MeAA-tRNAs to EF-Tu. This image was made from the cocrystal structure of Thermus aquaticus EF-Tu and Escherichia coli Phe-tRNA^Phe (PDB ID: 1TTT). The cross section around the binding pocket is shown in the upper panel. The hydrogen bond between EF-Tu Asn285 (blue) and Phe-tRNA^Phe (green) is indicated in yellow dotted line. (C) Uniform affinities of pAA-tRNAs to EF-Tu versus weak affinities of ^MeAA-tRNAs to EF-Tu. (D) Schematic representation of the affinity-tuning strategy of ^MeAAs and tRNAs demonstrated in this study.

Figure 2.

Weak affinities of npAA-tRNAs to EF-Tu correlating with the inaccurate translation of npAA-containing peptides. (A) The affinities of canonical pAA-tRNAs and npAA-tRNAs to EF-Tu. Asterisk (*) indicates undetectably weak affinity (ΔG > −6 kcal/mol). Error bar indicates the fitting error. (B) ^MeAAs and ϵ-N-acetyllysine used in this study. ^MeG, N-methylglycine; ^MeS, N-methylserine; ^MeA, N-methylalanine; ^MeF, N-methylphenylalanine; ^MeL, N-methylleucine; ^MeM, N-methylmethionine; ^MeT, N-methylthreonine; ^MeY, N-methyltyrosine; ^MeD, N-methylaspartic acid; ^MeV, N-methylvaline; ^MeNl, N-methylnorleucine; ^MeNv, N-methylnorvaline; ^MeYm, N-methyl-p-methoxyphenylalanine; and ^AcK, ϵ-N-acetyllysine. (C) Examination of the translation fidelity of peptides containing each of ^MeAAs or ^AcK. In each experiment, the mRNA1 containing GUC codon was translated in the FIT system containing npAA-tRNA^AsnE2_GAC of interest. Each dagger peak (†) corresponds to a byproduct containing Ile in place of npAA.

Figure 3.

Reinforcement of EF-Tu affinity of Phe-tRNA and npAA-tRNAs by T-stem engineering. (A) Sequence of the original tRNA^AsnE2 (#2) and T-stem variants #1–4. The affinities of tRNA #1–4 were designed to increase as the T-stem number increases from #1 to #4. (B–E) Quantification of the EF-Tu affinities of Phe-tRNA_GAC#1–4 (B, C) and ^MeF-tRNA_GAC#2–4 (D, E). Schematic representation of RNase A protection assay, the observed fraction of the ternary complex with the fitting curve to determine the K_D value (B, D), and the calculated ΔG value (C, E). Asterisk (*) indicates undetectably weak affinity (ΔG > −6 kcal/mol). Error bar indicates the fitting error. (F–J) The EF-Tu affinities of npAA-tRNA#1–4 and MALDI-TOF MS of P1-npAA examined for ^MeG (F), ^MeS (G), ^MeA (H), ^MeL (I) and ^AcK (J). Each dagger peak (†) corresponds to a byproduct containing Ile in place of npAA.

Figure 4.

Expression of an N-methyl-peptide containing 9 ^MeAAs and 15 pAAs. (A) Affinities of ^MeAA-tRNA#X to EF-Tu using all-#2, all-#4, and uniform sets. The orange bands indicate the range of ΔG values of pAA-tRNAs determined in Figure 2A (−8.0 to −9.4 kcal/mol). (B) Schematic representation of the EF-Tu affinity-tuning strategy. An appropriate combination of ^MeAA and tRNA#2–4 generates uniform affinities of ^MeAA-tRNA#Xs to EF-Tu comparable to canonical pAA-tRNAs. (C) The codon table reprogrammed with 9 ^MeAAs and 15 pAAs used in this study. (D) Sequences of mRNA2 and P2 peptide. (E) MALDI-TOF-MS analysis of P2 expressed using each set of ^MeAA and tRNA#X pairs. Arrowhead shows the corresponding monoisotopic peak of P2 or P2*. MALDI-TOF mass spectrum of P2-pAA is also shown.

Figure 5.

Expression of a macrocyclic peptide containing 9 ^MeAAs and 14 pAAs. (A) Sequences of mRNA3 and P3 peptide. (B) Structure of P3 closed via thioether bond. (C) MALDI-TOF MS analysis of P3 expressed with the uniformed set. Arrowhead shows the corresponding monoisotopic peak of P3.