Measuring the Mechanical Forces of Elongation Factor G Mediated Translocation



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The ribosome is the ribonucleoprotein complex that is responsible for the correct translation of mRNA into protein. This complex is associated with the GTPase elongation factor G (EF-G), which catalyzes translocation on the ribosome, however, the mechanism is poorly understood. Crystallographic studies revealed a previously unknown compact conformation of EF-G, implying large conformational changes. The biological relevance of this change was not revealed, however, because of the artificial crystal lattice present in x-ray studies. To explore this, an EF-G mutant carrying only two cysteines (M5 EF-G) was generated and internally crosslinked to itself, using two lengths of crosslinker. One that restricted EF-G from fully extending (BM(PEG)6) and one that allowed EF-G to undergo full conformational changes (BM(PEG)11). BM(PEG)11 crosslinked EF-G functioned comparably to wild type, while BM(PEG)6 crosslinked EF-G resulted in a lower force exerted. The BM(PEG)6 crosslinker also reduced ribosomal translocation efficiency to half, compared to that of the longer crosslinker and non-crosslinked EF-G. This force reduction did not result in frameshifting, confirming that EF-G does exert a force, but does not contribute to reading frame maintenance. Studying the overall conformational changes was important for understanding EF-G mechanism, however, it was also important to observe the details leading to the full conformational changes. In a second exploration of EF-G, the effector loop region was studied. The effector loop region spans approximately 7 residues within the GTPase center, the region where GTP is bound and hydrolyzed, on domain I of EF-G and contains important interactions for the function of EF-G. By introducing single site substitutions in the effector loop, the role of each residue was examined. Six variants were produced, and of the six, one was completely unable to hydrolyze GTP, while another exhibited reduced GTPase activity. Not a single of the six was able to promote translocation at a rate comparable to M5 EF-G, effectively abolishing the function of EF-G on the ribosome. By studying the effector loop of EF-G, further understanding of the roles of the residues within this region, and how they contribute to the overall function of EF-G was obtained.



EF-G, Ribosome, Translocation


Portions of this document appear in: Yin, H., Gavriliuc, M., Lin, R., Xu, S., & Wang, Y. (2019). Modulation and Visualization of EF‐G Power Stroke During Ribosomal Translocation. Chembiochem, 20(23), 2927-2935.