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

The ribosome translocates on the mRNA by primarily three nucleotides per step during protein synthesis, a process catalyzed by elongation factor G (EF-G). The large conformational changes of EF-G in this process generate significant mechanical force, referred to as power stroke. Quantification of power stroke remains under debate and its potential correlation with translocation fidelity has not been observed. In this work, we present a unique application of quantum sensing in combined measurements of the power strokes of mutated EF-G and their 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 a much reduced power stroke of 60 ± 6 pN and induced -1 frameshifting, wherein the ribosome translocated only two nucleotides on both sides of the mRNA. In contrast, Q508K, which interacts with tRNA residue 37 immediately outside the codon-anticodon minihelix, exhibited a power stroke of 89 ± 11 pN and maintained canonical 3-nt translocation, similar to wild-type EF-G. These findings provide direct mechanistic evidence that the pivotal point and force projection exerted by EF-G are critical for maintaining translocation fidelity, likely via lowering the kinetic energy barrier.