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Tertiary core rearrangements in a tight binding transfer RNA aptamer

Nature Structural Biology volume 7, pages 497–504 (2000)Cite this article

Abstract

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5′-44CAUUC48-3′ is replaced by 5′-44AGGU48-3′. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 Å cocrystal structure of the RNA aptamer–GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA–protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15–U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA–protein contacts.

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Figure 1: Structures of wild type and engineered tRNAs.
Figure 2: Electrophoretic gel mobility shift analysis for determination of the dissociation constant (Kd) of the var-AGGU engineered tRNA2Gln variant.
Figure 3: Stereo view showing difference electron density maps computed in the region of the variable loop of the var-AGGU mutant.
Figure 4: Structures of wild type and var-AGGU complexes.
Figure 5: van der Waals representation of bases in the tertiary core of the wild type (left) and var-AGGU (right) structures.
Figure 6: Overlay of mutant (black bonds) and wild type (gray bonds) tRNA structures in the region of the variable loop.
Figure 7: Plot of the average crystallographic thermal factor (B-factor) per nucleotide, as a function of nucleotide residue along the tRNA sequence.

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Acknowledgements

We thank O. Uhlenbeck and M. Derrick for encouragement and for communication of results prior to publication. We also thank B. Sproat and B. Beijer for the generous gift of QSI inhibitor. We are grateful to T. Earnest for assistance with data collection at ALS beamline 5.0.2, and to P. Allen for assistance with preparation of color figures. This work was supported by grants from the National Science Foundation (to J.J.P.) and from the Universitywide AIDS Research Program (to T.L.B.)

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  1. Department of Chemistry and Biochemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California at Santa Barbara, Santa Barbara, 93106-9510, California, USA

    Timothy L. Bullock, Luke D. Sherlin & John J. Perona

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  1. Timothy L. Bullock
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  2. Luke D. Sherlin
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  3. John J. Perona
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Correspondence to John J. Perona.

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Bullock, T., Sherlin, L. & Perona, J. Tertiary core rearrangements in a tight binding transfer RNA aptamer. Nat Struct Mol Biol 7, 497–504 (2000). https://doi.org/10.1038/75910

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