EF-G Mutations Reveal Correlation between Power Stroke and Translocation Fidelity in Protein Synthesis.

One of the most essential biomolecular functions is the ribosome translocation for protein synthesis, in which the ribosome moves on the mRNA by primarily three nucleotides per step in the presence of elongation factor G (EF-G). The large conformational changes of EF-G generate significant mechanical force, referred to as power stroke. Quantification of power stroke remains under debate and its correlation with translocation fidelity has not been observed. In this work, we use quantum sensing techniques for measuring both the EF-G power stroke and its influence on ribosome translocation steps. Two EF-G mutants, H584K and Q508K, were expressed, with the mutated residues directly interacting with tRNA. H584K, which interacts on codon-anticodon minihelix, produced less power stroke of 60±6 pN and induced "-1" frameshifting, wherein the ribosome translocated only two nucleotides. In contrast, Q508K, which interacts with tRNA residue 37 immediately outside the codon-anticodon minihelix, exhibited a normal power stroke of 89±11 pN and maintained canonical 3-nucleotide translocation. These findings provide direct mechanistic evidence that the pivotal point and EF-G power stroke are critical for maintaining translocation fidelity and highlight the potential of quantum sensing for chemical biology.