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Multi-channel smFRET study reveals a compact conformation of EF-G on the ribosome
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DOI：10.1016/j.biocel.2025.106782.png)
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Abstract
En 中文
While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-G's indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with eEF2's Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, the T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which was not observed when the mutants interact solely with the SRL. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.
Keywords:
Single molecule FRET
Multi-channel smFRET
Induced-fit
Ribosome translocation
Compact EF-G
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