Abstract
The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region. Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti -G56syn) and highly distorted backbones. The L30 protein is folded in a three-layer α/β/α sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop. The protein–RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10–U60 wobble base pair. Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation. The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA–protein interface is formed between regions on both molecules that lack regular secondary structure.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Burd, C.G. & Dreyfuss, G. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621 (1994).
Mager, W.H. et al. A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Nucleic Acids Res. 25, 4872–4875 (1997).
Dabeva, M.D. & Warner, J.R. The yeast ribosomal protein L32 and its gene. J. Biol. Chem. 262, 16055– 16059 (1987).
Eng, F.J. & Warner, J.R. Structural basis for the regulation of splicing of a yeast messenger RNA. Cell 65, 797–804 (1991).
Vilardell, J. & Warner, J.R. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes Dev. 8, 211–220 (1994).
Li, B., Vilardell, J. & Warner, J.R. An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness. Proc. Natl. Acad. Sci. USA 93, 1596– 1600 (1996).
Li, H., Dalal, S., Kohler, J., Vilardell, J. & White, S.A. Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop. J. Mol. Biol. 250, 447– 459 (1995).
Li, H. & White, S.A. RNA aptamers for yeast ribosomal protein L32 have a conserved purine-rich internal loop. RNA 3, 245–254 (1997).
Nagai, K. RNA–protein complexes. Curr. Opin. Struct. Biol. 6, 53–61 (1996).
Ramos, A., Gubser, C.C. & Varani, G. Recent solution structures of RNA and its complexes with drugs, peptides and proteins. Curr. Opin. Struct. Biol. 7, 317–323 (1997).
De Guzman, R.N. et al. Structure of the HIV-1 nucleocapsid protein bound to the SL3 Ψ-RNA recognition element. Science 279, 384– 388 (1998).
Price, S.R., Evans, P.R. & Nagai, K. Crystal structure of the spliceosomal U2B"–U2A' protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650 ( 1998).
Handa, N. et al. Structual basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature 398, 579– 585 (1999).
Conn, G.L., Draper, D.E., Lattman, E.E. & Gittis, A.G. Crystal structure of a conserved ribosomal protein–RNA complex. Science 284, 1171–1174 ( 1999).
Wimberly, B.T., Guymon, R., McCutcheon, J.P., White, S.W. & Ramakrishnan, V.A. Detailed view of a ribosomal active site: the structure of the L11–RNA complex. Cell 97, 491–502 (1999).
Mao, H. & Williamson, J.R. Assignment of the L30–mRNA complex using selective isotopic labeling and RNA mutants. Nucleic Acids Res. 27, 4059–4070 (1999).
Brünger, A.T. X-PLOR: a system for X-ray crystallography and NMR (Yale University Press, New Haven, Connecticut; 1992).
Cornell, W. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995).
Cate, J.H. et al. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678– 1685 (1996).
Ferre-D'Amare, A.R., Zhou, K. & Doudna, J. Crystal structure of a Hepatitis delta virus ribozyme. Nature 395, 567–574 (1998).
Stams, T., Niranjanakumari, S., Fierke, C.A. & Christianson, D.W. Ribonuclease P protein structure: evolutionary origins in the translational apparatus. Science 280, 752– 755 (1998).
Ramakrishnan, V. & White, S.W. The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA. Nature 358, 768–771 ( 1992).
Czworkowski, J., Wang, J., Steitz, T.A. & Moore, P.B. The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. EMBO. J. 13, 3661–3668 ( 1994).
Arnez, J.G. & Cavarelli, J. Structures of RNA-binding proteins. Q. Rev. Biophys. 30, 195– 240 (1997).
Mandel-Gutfreund, Y. & Margalit, H. Quantitative parameters for amino acid–base interaction: implications for prediction of protein–DNA binding site. Nucleic Acids Res. 26, 2306–2312. (1998).
White, S.A. & Li, H. Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition. RNA 2, 226– 234 (1995).
Szewczak, A.A. & Cech, T.R. An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis. RNA 3, 838–849 ( 1997).
Jiang, F., Kumar, R.A., Jones, R.A. & Patel, D.J. Structural basis of RNA folding and recognition in an AMP–RNA aptamer complex. Nature 382, 183–186 ( 1996).
Zimmermann, G.R., Jenison, R.D., Wick, C.L., Simorre, J.-P. & Pardi, A. Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA. Nature Struct. Biol. 4, 644–649 (1997).
Oubridge, C., Ito, N., Evans, P.R., Teo, C.H. & Nagai, K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438 ( 1994).
Allain, F.H.-T. et al. Specificity of ribonucleoprotein interaction determined by RNA folding during complex formation. Nature 380, 646–650 (1996).
Howe, P.W., Nagai, K., Neuhaus, D. & Varani, G. NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein. EMBO J. 13, 3873–3881 (1994).
Mao, H. & Williamson, J.R. Local folding coupled to RNA binding in the yeast ribosomal protein L30. J. Mol. Biol. 292, 345–359 (1999).
Markus, M.A., Hinck, A.P., Huang, S., Draper, D.E. & Torchia, D.A. High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that beocomes structured upon binding to RNA. Nature Struct. Biol. 4, 70–77 (1997).
Hinck, A.P. et al. The RNA binding domain of ribosomal protein L11: three-dimensional structure of the RNA-bound form of the protein and its interaction with 23S rRNA. J. Mol. Biol. 274, 101– 113 (1997).
Gubser, C.C. & Varani, G. Structure of the polyadenylation regulatory element of the human U1A pre-mRNA 3′-untranslated region and interaction with the U1A protein. Biochemistry 35, 2253–2267 (1996).
Allain, F.H., Howe, P.W., Neuhaus, D. & Varani, G. Structural basis of the RNA-binding specificity of human U1A protein. EMBO J. 16, 5764–5772 (1997).
Wyatt, J.R., Chastain, M. & Puglisi, J.D. Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11, 764– 769 (1991).
Batey, R.T., Inada, M., Kujawinski, E., Puglisi, J.D. & Williamson, J.R. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucleic Acids Res. 20, 4515–4523 (1992).
Ikura, M., Kay, L.E. & Bax, A. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29, 4659–4667 (1990).
Yamazaki, T., Lee, W., Arrowsmith, C.H., Muhandiram, D.R. & Kay, L.E. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J. Am. Chem. Soc. 116, 11655–11666 ( 1994).
Muhandiram, D.R. & Kay, L.E. Gradient-enhanced triple-resonance three-dimensional NMR experiments with improved sensitivity. J. Magn. Reson. B 103, 203– 216 (1994).
Kay, L.E., Keifer, P. & Saarinen, T. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114, 10663–10665 (1992).
Kay, L.E., Xu, G.-Y., Singer, A.U., Muhandiram, D.R. & Forman-Kay, J.D. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C correlations in H 2O samples of proteins. J. Magn. Reson. B 101 , 333–337 (1993).
Zuiderweg, E.R.P. & Fesik, S.W. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a. Biochemistry 28, 2387–2391 (1989).
Kumar, A., Ernst, R.R. & Wüthrich, K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem. Biophys. Res. Commun. 95, 1–6 (1980 ).
Rance, M. et al. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun. 117, 479–485 (1983).
Brauschweiler, L. & Ernst, R.R. Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J. Magn. Reson. 53, 521–528 (1983).
Bax, A. & Davis, D.G. Practical aspects of two-dimensional transverse NOE spectroscopy. J. Magn. Reson. 63, 207–213 (1985).
Mori, S., Abeygunawardana, C., Johnson, M.O. & van Zijl, P.C. Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J Magn Reson. B 108, 94– 98 (1995).
Bax, A., Ikura, M., Kay, L.E., Torchia, D.A. & Tschudin, R. Heteronuclear single quantum correlation. J. Magn. Reson. 86, 304–318 (1990).
Santoro, J. & King, G.C. A constant-time 2D Overbodenhausen experiment for inverse correlation of isotropically enriched species. J. Magn. Reson. 97, 202–207 (1992).
Pascal, S.M., Muhandiram, D.R., Yamazaki, T., Forman-Kay, J.D. & Kay, L.E. Simultaneous acquisition of 15N- and 13C-edited NOE spectra of proteins dissolved in H2O. J. Magn. Reson. B 103, 197–201 (1994).
Clore, G.M. & Gronenborn, A.M. Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. Science 252, 1390–1399 ( 1991).
Vuister, G.W. & Bax, A. Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins. J. Am. Chem. Soc. 115, 7772– 7777 (1993).
Kay, L.E., Brooks, B., Sparks, S.W., Tochia, D.A. & Bax, A. Measurement of NH-CαH coupling constants in staphylococcal nuclease by two-dimensional NMR and comparison with X-ray crystallographic results. J. Am. Chem. Soc. 111, 5488– 5490 (1989).
Clore, G.M., Bax, A. & Gronenborn, A.M. Stereospecific assignment of β-methylene protons in larger proteins using 3D 15N-separated Hartmann–Hahn and 13C-separated rotating frame Overhauser spectroscopy. J. Biomol. NMR 1, 13–22 (1991).
Garrett, D.S., Power, R., Gronenborn, A.M. & Clore, G.M. A commonsense approach to peak picking in two-, three-, and four-dimensional spectra using automatic computer analysis of contour diagrams. J. Magn. Reson. 95, 214–220 (1991).
Carson, M. Ribbon models of macromolecules. J. Mol. Graphics 5 , 103–106 (1987).
Acknowledgements
We would like to thank K. Dayie, C. Turner and J. Chung for assistance with NMR experiments, and R. Plachikkat and J. Schnell for valuable discussions in structural calculations. This work was supported by grants from the National Science Foundation to S.A.W. and the National Institutes of Health to J.R.W.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mao, H., White, S. & Williamson, J. A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex. Nat Struct Mol Biol 6, 1139–1147 (1999). https://doi.org/10.1038/70081
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/70081
This article is cited by
-
Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
BMC Molecular Biology (2013)
-
‘Popping the Clutch’: Novel Mechanisms Regulating Sexual Development in Cryptococcus neoformans
Mycopathologia (2012)
-
Size matters: a view of selenocysteine incorporation from the ribosome
Cellular and Molecular Life Sciences (2006)
-
Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes
Nature Structural & Molecular Biology (2005)
-
Localization and dynamic behavior of ribosomal protein L30e
Nature Structural & Molecular Biology (2005)