Synergy between eIF5A and Mg2+ enhances elongation in a defined yeast cell-free translation system with synthetic tRNAs

Cell-free translation platforms enable genetic-code engineering but typically rely on native transfer RNAs (tRNAs). Here we reconstitute Saccharomyces cerevisiae translation using a fully synthetic set of 21 in vitro-transcribed tRNAs—one isoacceptor per canonical amino acid plus the initiator—that collectively decode all 61 sense codons. After individual aminoacylation, this minimal pool supports peptide synthesis in a fully recombinant yeast cell-free translation system at yields comparable to native tRNAs. Translation of long open reading frames stalls without supplementation; adding either eukaryotic initiation factor 5A (eIF5A) or elevated Mg2+ restores activity, and together they increase Nano luciferase (NanoLuc) output by approximately fivefold. Alanine scanning implicates basic residues R27 and R87 in eIF5A, rather than the hypusine side chain, as the principal contributors to P-site tRNA stabilization. Higher Mg2+ alone accelerates elongation but increases UAG/UGA readthrough to ∼15%, revealing a tunable speed–fidelity trade-off with unmodified tRNAs. Installing t6A37 or m1G37 on selected synthetic tRNAs further improves processivity. This minimal, programmable yeast platform enables systematic dissection of tRNA-modification functions and provides a practical route to genetic-code engineering.