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

We reconstructed Saccharomyces cerevisiae translation with a fully synthetic panel of twenty-one in-vitro-transcribed tRNAs, one isoacceptor per canonical amino acid plus the initiator species. This minimal pool decodes all sixty-one sense codons and, after individual aminoacylation, drives peptide synthesis in a defined yeast PURE system at yields comparable to native tRNAs. Long peptides stall unless supplemented; adding either eukaryotic factor eIF5A or elevated Mg2+ restores activity, and their combination increases Nano luciferase output about fivefold. Alanine scanning shows that basic residues R27 and R87 within the eIF5A core, rather than the hypusine side chain, provide the principal P-site tRNA stabilization. Higher magnesium alone accelerates elongation but raises UAG and UGA read-through to roughly fifteen percent, revealing a trade-off between speed and fidelity when tRNAs are unmodified. Installing t6A37 or m1G37 on selected synthetic tRNAs further improves processivity. The resulting minimal yet programmable yeast platform enables systematic dissection of modification roles and rapid genetic-code engineering.本研究重建了酿酒酵母（Saccharomyces cerevisiae）的翻译体系，采用一套完全合成的21组分体外转录转运核糖核酸（in-vitro-transcribed tRNA）面板：每种标准氨基酸对应一种同功受体tRNA，外加起始型tRNA。该极简tRNA池可解码全部61个有义密码子；经单独氨酰化修饰后，该体系可在成分明确的酵母PURE无细胞蛋白合成系统（PURE system）中驱动肽链合成，其产率与天然tRNA相当。过长的肽链会出现合成停滞，需补充额外因子才能恢复；单独添加真核起始因子5A（eukaryotic initiation factor 5A, eIF5A）或提高镁离子（Mg²+）浓度均可恢复活性，二者联合使用可使纳米荧光素酶（Nano luciferase）的表达产量提升约5倍。丙氨酸扫描诱变实验显示，eIF5A核心结构域内的碱性残基R27与R87，而非羟腐胺赖氨酸（hypusine）侧链，是稳定P位转运RNA（P-site tRNA）的主要功能位点。单独提高镁离子浓度可加速肽链延伸，但会使UAG、UGA终止密码子的通读率提升至约15%，这揭示了未修饰tRNA在翻译速度与保真性之间存在权衡效应。在选定的合成tRNA上引入N6-苏氨酰基腺苷37（t6A37）或1-甲基鸟苷37（m1G37）修饰，可进一步提升翻译的持续合成能力。由此构建的极简且可程序化的酵母翻译平台，可用于系统性解析翻译修饰的功能，并支持快速的遗传密码工程改造。