Rotational Motions of Domains in Elongation Factor G Detected by Single-Molecule Polarized Fluorescence Microscopy

Abstract

During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule polarized total internal reflection fluorescence (polTIRF) microscopy to characterize tilting and rotational fluctuations within specific domains of EF-G. When EF-G binds to the ribosomal pre-translocation (PRE) complex, domains I and IV of EF-G undergo small rotations (10-15°) in conjunction with translocation, whereas domain III shows a much greater angular change, averaging 50°. Viomycin (Vio), which prevents translocation, reduces the rotational motions of domain III to 10-15° but has virtually no effect on the other domains. Spectinomycin also reduces domain III motions but less strongly than Vio. EF-G binding to ribosomal initiation complexes lacking A-site tRNA gives a similar pattern of domain rotations, but with shorter dwell times. In this case, the large rotation of domain III is barely inhibited by Vio. Irrespective of completion of translocation or presence of A-site tRNA, the initial 10-15° rotations of EF-G domains I, III and IV in the ribosome/EF-G complex indicate that the EF-G initially shifts the minimum of the free energy profile in the direction of translocation, suggesting that EF-G generates a force on the ribosome and/or the mRNA and tRNAs. Near the end of translocation, domain III completes its rotation either to push the mRNA and tRNAs (a working stroke) or to prevent reversal of translocation driven by thermal fluctuations (a ratchet). Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.