The ribosome derives the energy to translocate and unwind mRNA from EF-G binding

Sep 30, 2026

High-resolution optical tweezers combined with single-molecule fluorescence monitoring were used to follow EF-G binding to ribosomes together with two coupled processes: mRNA unwinding and mRNA translocation relative to the 30S body domain. EF-G mutants and GTP analogs show that neither unwinding nor translocation requires EF-G GTP hydrolysis, and the events can occur independently. Tight binding of EF-G is proposed to trigger both processes, while GTP hydrolysis accelerates translocation kinetics and is thermodynamically needed mainly to liberate EF-G from the ribosome.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages12

Reading Time30 min

AccessFree to download

Introduction

Mechanistic background of EF-G and translocation

Methods

Single-molecule optical tweezers with fluorescence detection

Results

EF-G GTP hydrolysis is not required for unwinding and translocation

Independence of unwinding and translocation

Model and implications

Tight binding triggers both processes; hydrolysis required for EF-G release

Article [https://doi.org/10.1038/s41467-025-66812-7](https://doi.org/10.1038/s41467-025-66812-7) The ribosome derives the energy to translocate and unwind mRNA from EF-G binding | Received: 7 January 2025
Accepted: 17 November 2025
| Hossein Amiri 1,2,3,4,11 , William J. Van Patten1,2,5,11, Gillian Rexroad6,7, Varsha P. Desai1,2, Benjamen A. Sterwerf1,2, Laura Lancaster6,7,
Harry F. Noller6,7 & Carlos Bustamante 1,2,4,5,8,9,10 | | | --- | --- | --- | | | The GTPase EF-G catalyzes translocation of mRNA and tRNAs relative to the ribosome and helps maintain the reading frame during protein synthesis. Which events directly require EF-G-mediated GTP hydrolysis during translocation are still debated. Using high-resolution optical tweezers endowed with single-molecule ﬂuorescence detection, we simultaneously monitored binding of ﬂuorescently-labeled EF-G to ribosomes and either mRNA unwinding or mRNA translocation relative to the body domain of the small ribosomal subunit. Using EF-G mutants and GTP analogs, we ﬁnd that neither mRNA unwinding nor translocation require GTP hydrolysis and that these are independent events that may or may not temporally coincide. We propose that“tight binding” of EF-G to the ribosome triggers mRNA unwinding and translocation of mRNA relative to the 30S body domain and that while GTP hydrolysis kinetically accelerates translocation, it is thermodynamically required only to liberate the tightly bound EF-G from the ribosome. | | | During the elongation phase of protein synthesis, the ribosome translocates mRNA in single-codon (3 nt) steps1,2. This coordinated translocation of the mRNA and tRNAs is catalyzed by the GTPase elongation factor EF-G and guided by conformational changes of the large and small ribosomal subunits following peptide bond formation3–10. Speciﬁcally, the 30S subunit rotates with respect to the 50S subunit, which moves the tRNAs on the 50S subunit resulting in hybrid tRNA states11. Next, forward rotation or “swivel” of the 30S head domain, accompanied by a partial reversion of inter-subunit rotation, moves the mRNA and tRNAs relative to the body domain to produce chimeric hybrid tRNA states12,13. This step is followed by the reverse 30S head rotation without carrying back the mRNA and tRNAs, which together with full reverse inter-subunit rotation, completes the mRNA/ | | tRNA movement and resets the ribosome to the classical posttranslocation state.
Translocation involves directional mRNA movement in precise codon steps and unwinding of mRNA secondary structures2,7,14. In the absence of an external energy source, these processes are endergonic (ΔG > 0) and will not occur spontaneously. To drive these processes, the ribosome must couple them with exergonic reactions, which could include peptidyl transfer between P- and A-site tRNAs and EF-Gmediated GTP hydrolysis. After GTP hydrolysis, EF-G retains the cleaved γ-phosphate for some time15,16 before releasing it as inorganic phosphate (Pi), which contributes to the free energy available from hydrolysis. Two roles for GTP hydrolysis and Pi release by EF-G during translocation have been described. In the ﬁrst role, the energy from | 1Institute for Quantitative Biosciences-QB3, University of California, Berkeley, CA, USA. 2Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA, USA. 3Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. 4Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. 5Biophysics Graduate Group, University of California, Berkeley, CA, USA. 6Center for Molecular Biology of RNA, University of California, Santa Cruz, CA, USA. 7Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA, USA. 8Department of Chemistry, University of California, Berkeley, CA, USA. 9Department of Physics, University of California, Berkeley, CA, USA. 10Kavli Energy Nanoscience Institute, U

FAQs

What was measured in the experiments on ribosome translocation?

The study simultaneously monitored binding of fluorescently labeled EF-G to ribosomes and either mRNA unwinding or mRNA translocation relative to the 30S body domain.

Does mRNA unwinding during translocation require EF-G-mediated GTP hydrolysis?

No. Using EF-G mutants and GTP analogs, the experiments show that mRNA unwinding does not require GTP hydrolysis.

What role does GTP hydrolysis play according to the proposed model?

GTP hydrolysis accelerates translocation kinetics, but it is thermodynamically required mainly to liberate tightly bound EF-G from the ribosome.

Sep 30, 2026

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