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

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Elongation factor G (EF-G) is essential for bacterial ribosome translocation.
• The precise role of GTP hydrolysis in EF-G function is not fully understood.
• Phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56 inhibits protein synthesis, but the mechanism is unclear.

Purpose of the Study:

• To investigate the conformational dynamics of E. coli EF-G using single-molecule Förster Resonance Energy Transfer (smFRET).
• To explore the impact of mutations mimicking eEF2 phosphorylation on EF-G function and conformation.
• To elucidate the mechanism of EF-G-mediated ribosome translocation and its regulation.

Main Methods:

• Development of a multi-channel smFRET microscopy technique.
• Utilized double-labeled EF-G (Alexa 488/594) and ribosome-bound tRNA (Cy3) and ribosomal protein L27 (Cy5).
• Analyzed conformational changes of EF-G during the translocation of fMet-Phe-tRNA(Phe) within labeled ribosomes.

Main Results:

• Wild-type EF-G adopts an extended conformation upon ribosome binding with GTP or GDPCP, facilitating translocation.
• Mutant EF-G variants (T48E, T48V) retained GTP binding and hydrolysis but exhibited impaired Poly(Phe) synthesis.
• These mutants adopted a compact conformation, distinct from interactions with the sarcin/ricin loop alone, hindering translocation.

Conclusions:

• EF-G exhibits conformational adaptability crucial for its function in ribosome translocation.
• The study provides mechanistic insights into how eEF2 modification impacts protein synthesis.
• The findings highlight the importance of specific EF-G conformations for efficient protein synthesis.