Coordinated conformational and compositional dynamics drive ribosome translocation

• Published in: Nature structural & molecular biology Vol. 20; no. 6; pp. 718 - 727
• Main Authors: Chen, Jin, Petrov, Alexey, Tsai, Albert, O'Leary, Seán E, Puglisi, Joseph D
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.06.2013; Nature Publishing Group
• Subjects: 631/337/574/1789; 631/57/2265; 631/57/2272/1590; Biochemistry; Biological Microscopy; Biosynthesis; E coli; Escherichia coli - chemistry; Escherichia coli - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate - metabolism; Hydrolysis; Life Sciences; Membrane Biology; Models, Biological; Molecular biology; Molecular dynamics; Peptide Elongation Factor G - metabolism; Properties; Protein Biosynthesis; Protein Conformation; Protein Structure; Protein synthesis; Ribonucleic acid; Ribosomes; Ribosomes - chemistry; Ribosomes - metabolism; RNA; RNA, Transfer - metabolism; Translocation; Translocation (Genetics)
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.2567

During translation, elongation factor G (EF-G) and transfer RNAs alternately bind the ribosome to direct protein synthesis. Using single-molecule fluorescence with zero-mode waveguide, EF-G–GTP is shown to continuously sample both rotational states of the ribosome, binding with higher affinity to the rotated state to stimulate translocation and return to the nonrotated state. During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA ) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli . Our results show that EF-G bound to GTP (EF-G–GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G–ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G–GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.