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Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p

Nature Structural & Molecular Biology volume 15, pages 397–402 (2008)Cite this article

Abstract

Pumilio/FBF (PUF) family proteins are found in eukaryotic organisms and regulate gene expression post-transcriptionally by binding to sequences in the 3′ untranslated region of target transcripts. PUF proteins contain an RNA binding domain that typically comprises eight α-helical repeats, each of which recognizes one RNA base. Some PUF proteins, including yeast Puf4p, have altered RNA binding specificity and use their eight repeats to bind to RNA sequences with nine or ten bases. Here we report the crystal structures of Puf4p alone and in complex with a 9-nucleotide (nt) target RNA sequence, revealing that Puf4p accommodates an 'extra' nucleotide by modest adaptations allowing one base to be turned away from the RNA binding surface. Using structural information and sequence comparisons, we created a mutant Puf4p protein that preferentially binds to an 8-nt target RNA sequence over a 9-nt sequence and restores binding of each protein repeat to one RNA base.

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Figure 1: Crystal structure of the Puf4p RNA binding domain.
Figure 2: Recognition of RNA by the Puf4p RNA binding domain.
Figure 3: Crystal structure of the mutPuf4p–cox17 RNA complex.

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Acknowledgements

We thank K. Adelman and L. Pedersen for critical comments on the manuscript. We are grateful to A. Sigova of the University of Massachusetts Medical School for the yeast genomic DNA, A. Clark for advice on purifying yeast genomic DNA, J. Holmes for technical assistance, L. Pedersen for crystallography support and D. Esposito of the Protein Expression Lab, NCI-Frederick, for the pDEST-565 plasmid. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. The Advanced Photon Source used for this study was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We thank Z. Jin for collecting these data at SER-CAT as part of the mail-in crystallography program.

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  1. Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Drive, Building 101, Room F363, Research Triangle Park, 27709, North Carolina, USA

    Matthew T Miller, Joshua J Higgin & Traci M Tanaka Hall

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  1. Matthew T Miller
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Contributions

J.J.H., M.T.M. and T.M.T.H. designed the experiments; J.J.H. crystallized native Puf4p; M.T.M. determined the structures of native and mutant Puf4p and performed the RNA binding analyses; M.T.M. and T.M.T.H. prepared the manuscript.

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Correspondence to Traci M Tanaka Hall.

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Miller, M., Higgin, J. & Tanaka Hall, T. Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p. Nat Struct Mol Biol 15, 397–402 (2008). https://doi.org/10.1038/nsmb.1390

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