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Science 20 December 1996:
Vol. 274. no. 5295, pp. 2065 - 2069
DOI: 10.1126/science.274.5295.2065
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Reports

Capturing the Structure of a Catalytic RNA Intermediate: The Hammerhead Ribozyme

William G. Scott, * James B. Murray, John R. P. Arnold, Barry L. Stoddard, Aaron Klug

The crystal structure of an unmodified hammerhead RNA in the absence of divalent metal ions has been solved, and it was shown that this ribozyme can cleave itself in the crystal when divalent metal ions are added. This biologically active RNA fold is the same as that found previously for two modified hammerhead ribozymes. Addition of divalent cations at low pH makes it possible to capture the uncleaved RNA in metal-bound form. A conformational intermediate, having an additional Mg(II) bound to the cleavage-site phosphate, was captured by freeze-trapping the RNA at an active pH prior to cleavage. The most significant conformational changes were limited to the active site of the ribozyme, and the changed conformation requires only small additional movements to reach a proposed transition-state.

W. G. Scott and A. Klug, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
J. B. Murray and J.R.P. Arnold, Department of Biology, University of Leeds, Leeds LS2 9JT, England.
B. L. Stoddard, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104, USA.
*   To whom correspondence should be addressed.


References

  • AMIRI, KMA, The global conformation of an active hammerhead RNA during the process of self-cleavage, JOURNAL OF MOLECULAR BIOLOGY 261: 125 (1996).
  • BASSI, G.S., IONIC INTERACTIONS AND THE GLOBAL CONFORMATIONS OF THE HAMMERHEAD RIBOZYME, NATURE STRUCTURAL BIOLOGY 2: 45 (1995).
  • BOLDUC, J.M., MUTAGENESIS AND LAUE STRUCTURES OF ENZYME INTERMEDIATES - ISOCITRATE DEHYDROGENASE, SCIENCE 268: 1312 (1995).
  • BRUNGER, A.T., XPLOR 3 1 SYSTEM CRY (1993).
  • DAHM, S, BIOCHEMISTRY-US 32: 13010 (1993).
  • DAHM, S.C., ROLE OF DIVALENT METAL-IONS IN THE HAMMERHEAD RNA CLEAVAGE REACTION, BIOCHEMISTRY 30: 9464 (1991).
  • GUERRIERTAKADA, C, THE RNA MOIETY OF RIBONUCLEASE-P IS THE CATALYTIC SUBUNIT OF THE ENZYME, CELL 35: 849 (1983).
  • HERTEL, K.J., THE INTERNAL EQUILIBRIUM OF THE HAMMERHEAD RIBOZYME REACTION, BIOCHEMISTRY 34: 1744 (1995).
  • KOIZUMI, M, BIOCHEMISTRY-US 30: 145 (1991).
  • MCKAY, D.B., Structure and function of the hammerhead ribozyme: An unfinished story, RNA-A PUBLICATION OF THE RNA SOCIETY 2: 395 (1996).
  • MOFFAT, K, FREEZE TRAPPING OF REACTION INTERMEDIATES, CURRENT OPINION IN STRUCTURAL BIOLOGY 5: 656 (1995).
  • MURRAY, J.B., A GENERAL PURIFICATION PROCEDURE FOR CHEMICALLY SYNTHESIZED OLIGORIBONUCLEOTIDES, ANALYTICAL BIOCHEMISTRY 218: 177 (1994).
  • MURRAY, J.B., THE ROLES OF THE CONSERVED PYRIMIDINE-BASES IN HAMMERHEAD RIBOZYME CATALYSIS - EVIDENCE FOR A MAGNESIUM ION-BINDING SITE, BIOCHEMICAL JOURNAL 311: 487 (1995).
  • OTWINOWSKI, Z, P CCP4 STUD WEEK DAT 56 (1993).
  • PARKINSON, G, New parameters for the refinement of nucleic acid-containing structures, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 52: 57 (1996).
  • PLEY, H.W., MODEL FOR AN RNA TERTIARY INTERACTION FRONT THE STRUCTURE OF AN INTERMOLECULAR COMPLEX BETWEEN A GAAA TETRALOOP AND AN RNA HELIX, NATURE 372: 111 (1994).
  • RUFFNER, D.E., SEQUENCE REQUIREMENTS OF THE HAMMERHEAD RNA SELF-CLEAVAGE REACTION, BIOCHEMISTRY 29: 10695 (1990).
  • SCOTT, W.G., Ribozymes: Structure and mechanism in RNA catalysis, TRENDS IN BIOCHEMICAL SCIENCES 21: 220 (1996).
  • SCOTT, W.G., THE CRYSTAL-STRUCTURE OF AN ALL-RNA HAMMERHEAD RIBOZYME - A PROPOSED MECHANISM FOR RNA CATALYTIC CLEAVAGE, CELL 81: 991 (1995).
  • SCOTT, W.G., RAPID CRYSTALLIZATION OF CHEMICALLY SYNTHESIZED HAMMERHEAD RNAS USING A DOUBLE SCREENING-PROCEDURE, JOURNAL OF MOLECULAR BIOLOGY 250: 327 (1995).
  • SIGURDSSON, S.T., PROBING RNA TERTIARY STRUCTURE - INTERHELICAL CROSS-LINKING OF THE HAMMERHEAD RIBOZYME, RNA-A PUBLICATION OF THE RNA SOCIETY 1: 575 (1995).
  • SLIM, G, CONFIGURATIONALLY DEFINED PHOSPHOROTHIOATE-CONTAINING OLIGORIBONUCLEOTIDES IN THE STUDY OF THE MECHANISM OF CLEAVAGE OF HAMMERHEAD RIBOZYMES, NUCLEIC ACIDS RESEARCH 19: 1183 (1991).
  • TUSHL, T, SCIENCE 266: 785 (1994).
  • UHLENBECK, O.C., A SMALL CATALYTIC OLIGORIBONUCLEOTIDE, NATURE 328: 596 (1987).
  • VANTOL, H, 2 AUTOLYTIC PROCESSING REACTIONS OF A SATELLITE RNA PROCEED WITH INVERSION OF CONFIGURATION, NUCLEIC ACIDS RESEARCH 18: 1971 (1990).
  • YANG, J.H., MIXED DNA RNA POLYMERS ARE CLEAVED BY THE HAMMERHEAD RIBOZYME, BIOCHEMISTRY 29: 11156 (1990).
  • ZAUG, A.J., THE INTERVENING SEQUENCE RNA OF TETRAHYMENA IS AN ENZYME, SCIENCE 231: 470 (1986).


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