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Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation

Nature Chemical Biology volume 6, pages 344–351 (2010)Cite this article

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Abstract

Following ribosomal peptide bond formation, the reaction products, peptidyl-tRNA and deacylated tRNA, need to be translocated from the A- and P-sites to the P- and E-sites, respectively. This process is facilitated by the GTPase elongation factor G (EF-G). The mechanism describing how the ribosome activates GTP hydrolysis is poorly understood in molecular terms. By using an 'atomic mutagenesis' approach, which allows the manipulation of specific functional groups on 23S rRNA nucleotides in the context of the entire ribosome, we disclose the adenine exocyclic N6 amino group at A2660 of the sarcin-ricin loop as a key determinant for triggering GTP hydrolysis on EF-G. We show that the purine π system–expanding characteristics of the exocyclic functional group at the C6 position of A2660 are essential. We propose that stacking interactions of A2660 with EF-G may act as a molecular trigger to induce repositioning of suspected functional amino acids in EF-G that in turn promote GTP hydrolysis.

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Figure 1: 23S rRNA constructs and uncoupled EF-G GTPase activities of wild-type ribosomes.
Figure 2: EF-G GTPase activities using reconstituted ribosomes.
Figure 3: Atomic mutagenesis at A2660 of the SRL.
Figure 4: Binding activity of EF-G to reconstituted ribosomes.

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Acknowledgements

We thank Y.-L. Chan, S. Connell, M. Erlacher, M. Pech, P. Sergiev, T. Tenson, O. Vesper, D. Wilson and M. Zywicki for valuable comments and suggestions. A. Hüttenhofer is acknowledged for his support at the Division of Genomics and RNomics. We thank A. Mankin (University of Illinois at Chicago) and N. Shankaran (University of Illinois at Chicago) for the GAC deletion mutant, M. Sprinzl (University of Bayreuth) for the parental EF-G clone, T. Tenson (University of Tartu) for the LepA clone and W. Piendl and G. Brosch for help with the EF-G and LepA purifications, respectively. We acknowledge F. Handle for experimental help during a lab rotation. This work was funded by the Austrian Science Foundation (FWF) (I317 and P21641 to R.M. and Y315 to N.P.) and the Austrian Ministry of Science and Research (GenAU project consortium 'non-coding RNAs' P0726-012-012 to R.M. and D-110420-012-012 to N.P.).

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Authors and Affiliations

  1. Division of Genomics and RNomics, Innsbruck Biocenter, Medical University Innsbruck, Innsbruck, Austria

    Nina Clementi, Anna Chirkova & Norbert Polacek

  2. Institute of Organic Chemistry, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria

    Barbara Puffer & Ronald Micura

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  1. Nina Clementi
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Contributions

N.C. established the GTPase and EF-G binding assays with reconstituted ribosomes and contributed to all figures. A.C. optimized the in vitro translation assay and contributed to Figure 3 and Supplementary Figure 2. B.P. synthesized non-natural nucleoside analogs under the supervision of R.M. N.P., R.M. and N.C. analyzed the data. N.P. designed the study and wrote the paper (together with R.M. and N.C.).

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Correspondence to Norbert Polacek.

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Clementi, N., Chirkova, A., Puffer, B. et al. Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation. Nat Chem Biol 6, 344–351 (2010). https://doi.org/10.1038/nchembio.341

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