Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

Abstract

While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. The indispensability of EF-G 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 Thr56 residue of eEF2. 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, 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 sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.

Developing Multichannel smFRET Approach to Dissecting Ribosomal Mechanisms

“…These findings, though derived from an initiation complex that cannot translocate, can be extrapolated to normal pretranslocation complexes, suggesting a mechanism by which EF-G facilitates translocation. Additional biological insights from this method have been reported recently, offering further understanding of the intricate dynamics involved in ribosome function …”

“…Additional biological insights from this method have been reported recently, offering further understanding of the intricate dynamics involved in ribosome function. 38 …”

“…Chemicals are from Millipore-Sigma if not specified. More detailed biological preparations are described in ref ( 38 ). The details of biomolecule preparation are in Supporting Information .…”