GTP hydrolysis by EF‐G synchronizes tRNA movement on small and large ribosomal subunits

• Published in: The EMBO journal 2014-05, Vol.33 (9), p.1073-1085
• Main Authors: Holtkamp, Wolf, Cunha, Carlos E, Peske, Frank, Konevega, Andrey L, Wintermeyer, Wolfgang, Rodnina, Marina V
• Format: Article
• Language: English
• Subjects: Antibiotics; Bacteriology; EMBO32; Escherichia coli - genetics; Escherichia coli - metabolism; Guanosine Triphosphate - metabolism; Hydrolysis; Kinetics; molecular machines; Movement; Organisms, Genetically Modified; Peptide Elongation Factor G - metabolism; Protein Biosynthesis; protein synthesis; Ribosomal DNA; ribosome; Ribosome Subunits, Large, Bacterial - metabolism; Ribosome Subunits, Small, Bacterial - metabolism; RNA Transport; RNA, Transfer - physiology; translation; Translocation
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/embj.201387465

Elongation factor G (EF‐G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF‐G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF‐G mutants and translocation‐specific antibiotics to investigate timing and energetics of translocation. We show that EF‐G–GTP facilitates synchronous movements of peptidyl‐tRNA on the two subunits into an early post‐translocation state, which resembles a chimeric state identified by structural studies. EF‐G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF‐G. Our results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit. Synopsis A new dual‐color labeling approach yields dynamic insight on synchronous tRNA movement on both ribosomal subunits and shows that GTP hydrolysis by the translocation factor EF‐G drives rearrangement of the 30S subunit. Rapid translocation promoted by EF‐G‐GTP proceeds synchronously on both ribosomal subunits. Synchronization of translocation is mediated through the 30S subunit. Rapid 30S translocation requires GTP hydrolysis and movement of domain 4 of EF‐G. 50S translocation entails a transient intermediate distinct from the hybrid state. Graphical Abstract A new dual‐color labeling approach yields dynamic insight on synchronous tRNA movement on both ribosomal subunits and shows that GTP hydrolysis by the translocation factor EF‐G drives rearrangement of the 30S subunit.