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Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme

Nature volume 454, pages 358–361 (2008)Cite this article

Abstract

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro1,2,3,4,5. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3′-OH5,6,7. Here we report the 2.8-Å resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases8,9 to increase specificity.

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Figure 1: Overall structure of the flexizyme–tRNA minihelix fusion.
Figure 2: Comparison of protein and RNA aminoacyl-tRNA synthetases.
Figure 3: Structure and sequence requirements of the active site.
Figure 4: Conjectural coupling of tRNA docking to active-site folding.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes for aminoacyl-tRNA synthetase ribozyme–minihelix fusions refined against crystal II and crystal III data have been deposited with the Protein Data Bank with accession codes 3CUL and 3CUN, respectively.

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Acknowledgements

We thank the staff at ALS beamline 5.0.2 and J. Bolduc for assistance with synchrotron and in-house X-ray data collection, respectively, and T. Edwards, T. Hamma, D. Klein, J. Pitt, J. Posakony, A. Roll-Mecak and B. Shen for discussions. A.R.F. is a Distinguished Young Scholar in Medical Research of the W. M. Keck Foundation. This work was supported by grants from research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization (to H.S.) and the W. M. Keck Foundation (to A.R.F.).

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Authors and Affiliations

  1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109-1024, USA,

    Hong Xiao & Adrian R. Ferré-D’Amaré

  2. Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan

    Hiroshi Murakami & Hiroaki Suga

  3. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 113-8656, Tokyo, Japan,

    Hiroaki Suga

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  1. Hong Xiao
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Corresponding author

Correspondence to Adrian R. Ferré-D’Amaré.

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The file contains Supplementary Figures 1-8 with Legends, Supplementary Table 1 and additional references. (PDF 1067 kb)

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Xiao, H., Murakami, H., Suga, H. et al. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358–361 (2008). https://doi.org/10.1038/nature07033

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