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

Abstract

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.