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The hybrid state of tRNA binding is an authentic translation elongation intermediate

Nature Structural & Molecular Biology volume 13, pages 234–241 (2006)Cite this article

Abstract

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA–transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre–steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.

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Figure 1: Schematic drawing of relationship between pre- and post-translocation state ribosomes and of known rRNA–tRNA pairing interactions.
Figure 2: Toeprinting analysis of sparsomycin- and EF-G–mediated translocation on m301 messenger RNA.
Figure 3: Analysis of sparsomycin-mediated translocation and puromycin reactivity of pretranslocation ribosome complexes carrying initiator and elongator tRNAMet.
Figure 4: Pre–steady-state kinetic analysis of EF-G–mediated translocation.

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Acknowledgements

We thank C. Merryman and other members of the lab for discussions, J. Lorsch (Johns Hopkins University) for critical reading of the manuscript and support with our kinetic studies on the ribosome, K. Fredrick (The Ohio State University) for the m301 plasmid, S. Blanchard (Cornell University) for the EF-G construct and O. Uhlenbeck (Northwestern University) for plasmid p67CF10. The work was supported by an Erwin Schrödinger fellowship (J2172) from the Austrian Science Foundation to S.D., by the US National Institutes of Health and by salary support from the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Silke Dorner, Julie L Brunelle, Divya Sharma & Rachel Green

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  2. Julie L Brunelle
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Correspondence to Rachel Green.

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Supplementary information

Supplementary Fig. 1

Sparsomycin-mediated translocation of G2251 and G2252 mutant ribosomes. (PDF 272 kb)

Supplementary Fig. 2

Comparison of native and transcribed initiator tRNAs. (PDF 106 kb)

Supplementary Fig. 3

FPLC analysis of GDP contamination in GTP. (PDF 135 kb)

Supplementary Fig. 4

K1/2 for EF-G on various ribosomal complexes. (PDF 90 kb)

Supplementary Table 1

Hybrid reactivity of dipeptidyl tRNA complexes with puromycin (PDF 68 kb)

Supplementary Table 2

EF-G dependent translocation with GTP, GTP or GTP/GDP (PDF 67 kb)

Supplementary Table 3

EF-G dependent translocation with/without energy regeneration system (PDF 82 kb)

Supplementary Methods

Primer for PCR of tRNAs (PDF 72 kb)

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Dorner, S., Brunelle, J., Sharma, D. et al. The hybrid state of tRNA binding is an authentic translation elongation intermediate. Nat Struct Mol Biol 13, 234–241 (2006). https://doi.org/10.1038/nsmb1060

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