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PMID: 10535916 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Three metal ions at the active site of the Tetrahymena group I ribozyme.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 96 ·No. 22 ·1999-10-26 ·Pages 12299-304

Shan So, Yoshida A, Sun S, Piccirilli JA, Herschlag D

Abstract

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3'-oxygen of the oligonucleotide substrate and the 3'- and 2'-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

MeSH Terms
Animals Binding Sites Catalysis Kinetics Metals/chemistry RNA, Catalytic/chemistry,metabolism Tetrahymena/enzymology
Chemicals
Metals RNA, Catalytic
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Shan S o
Department of Biochemistry, Stanford University, Stanford, CA 94305-5307, USA.
Yoshida A
Sun S
Piccirilli J A
Herschlag D
References (63)
63 references, click to expand
  1. Contributions of 2'-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme. An energetic picture of an active site composed of RNA.
    Biochemistry. 1993 Aug 17;32(32):8299-311 PMID: 7688572
  2. Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor.
    Biochemistry. 1986 Aug 12;25(16):4473-7 PMID: 3639741
  3. Role of metal ions in ribozymes.
    Met Ions Biol Syst. 1996;32:479-520 PMID: 8640529
  4. Probing the role of metal ions in RNA catalysis: kinetic and thermodynamic characterization of a metal ion interaction with the 2'-moiety of the guanosine nucleophile in the Tetrahymena group I ribozyme.
    Biochemistry. 1999 Aug 24;38(34):10958-75 PMID: 10460151
  5. Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
    Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13489-94 PMID: 9811827
  6. Crystallographic studies of the catalytic mechanism of the neutral form of fructose-1,6-bisphosphatase.
    Biochemistry. 1993 Feb 23;32(7):1844-57 PMID: 8382525
  7. Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding.
    Biochemistry. 1986 Oct 21;25(21):6365-71 PMID: 3539189
  8. A minor groove RNA triple helix within the catalytic core of a group I intron.
    Nat Struct Biol. 1998 Dec;5(12):1037-42 PMID: 9846872
  9. X-ray structure and catalytic mechanism of lobster enolase.
    Biochemistry. 1995 Oct 3;34(39):12513-23 PMID: 7547999
  10. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.
    Nature. 1991 Apr 18;350(6319):628-31 PMID: 1708111
  11. Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.
    Nucleic Acids Res. 1989 Jan 11;17(1):355-71 PMID: 2643083
  12. Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.
    Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8924-8 PMID: 8799129
  13. Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.
    Biochemistry. 1997 Mar 4;36(9):2465-77 PMID: 9054551
  14. A mechanism for all polymerases.
    Nature. 1998 Jan 15;391(6664):231-2 PMID: 9440683
  15. Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond.
    Biochemistry. 1997 Mar 4;36(9):2425-38 PMID: 9054547
  16. Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme.
    Biochemistry. 1993 Dec 14;32(49):13593-604 PMID: 7504953
  17. Ribozyme-mediated cleavage of a substrate analogue containing an internucleotide-bridging 5'-phosphorothioate: evidence for the single-metal model.
    Biochemistry. 1996 Apr 23;35(16):5308-17 PMID: 8611518
  18. Activation/attenuation model for RNase H. A one-metal mechanism with second-metal inhibition.
    J Biol Chem. 1998 Dec 18;273(51):34128-33 PMID: 9852071
  19. Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization.
    Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3668-72 PMID: 7731962
  20. Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.
    RNA. 1997 Nov;3(11):1352-63 PMID: 9409625
  21. Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site.
    Science. 1995 Feb 3;267(5198):675-9 PMID: 7839142
  22. Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.
    Nucleic Acids Res. 1997 Sep 1;25(17):3421-7 PMID: 9254698
  23. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution.
    Biochemistry. 1995 Jan 17;34(2):683-96 PMID: 7819264
  24. A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme.
    Chem Biol. 1999 Mar;6(3):153-65 PMID: 10074469
  25. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods.
    Science. 1992 Nov 20;258(5086):1355-8 PMID: 1455230
  26. Metal ion catalysis in the Tetrahymena ribozyme reaction.
    Nature. 1993 Jan 7;361(6407):85-8 PMID: 8421499
  27. The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction.
    Biochemistry. 1993 Aug 17;32(32):8312-21 PMID: 7688573
  28. A magnesium ion core at the heart of a ribozyme domain.
    Nat Struct Biol. 1997 Jul;4(7):553-8 PMID: 9228948
  29. Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2+-phosphonoacetohydroxamate complex at 2.4-A resolution.
    Biochemistry. 1994 May 24;33(20):6295-300 PMID: 8193144
  30. Strategies for RNA folding.
    Trends Biochem Sci. 1996 Apr;21(4):145-9 PMID: 8701472
  31. A general two-metal-ion mechanism for catalytic RNA.
    Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498-502 PMID: 8341661
  32. Effects of divalent metal ions on individual steps of the Tetrahymena ribozyme reaction.
    Biochemistry. 1997 Jul 8;36(27):8293-303 PMID: 9204875
  33. Protonated 2'-aminoguanosine as a probe of the electrostatic environment of the active site of the Tetrahymena group I ribozyme.
    Biochemistry. 1999 Aug 24;38(34):10976-88 PMID: 10460152
  34. Binuclear Metallohydrolases.
    Chem Rev. 1996 Nov 7;96(7):2435-2458 PMID: 11848832
  35. Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila.
    Biochemistry. 1987 Jun 16;26(12):3330-40 PMID: 2443161
  36. Dissection of the role of the conserved G.U pair in group I RNA self-splicing.
    Biochemistry. 1994 Nov 22;33(46):13864-79 PMID: 7947795
  37. A re-investigation of the thio effect at the hammerhead cleavage site.
    Nucleic Acids Res. 1999 Jan 15;27(2):479-84 PMID: 9862968
  38. How many catalytic RNAs? Ions and the Cheshire cat conjecture.
    FASEB J. 1993 Jan;7(1):31-9 PMID: 8422972
  39. Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.
    Biochemistry. 1988 Dec 13;27(25):8924-31 PMID: 3069131
  40. The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations.
    Structure. 1996 Jan 15;4(1):89-96 PMID: 8805516
  41. The environment of two metal ions surrounding the splice site of a group I intron.
    EMBO J. 1996 May 15;15(10):2556-64 PMID: 8665863
  42. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site.
    Biochemistry. 1990 Nov 6;29(44):10159-71 PMID: 2271645
  43. Multiple magnesium ions in the ribonuclease P reaction mechanism.
    Biochemistry. 1993 May 25;32(20):5273-81 PMID: 8499432
  44. A positive entropy change for guanosine binding and for the chemical step in the Tetrahymena ribozyme reaction.
    Biochemistry. 1995 Mar 28;34(12):4056-67 PMID: 7696271
  45. Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp.
    Biochemistry. 1996 Aug 13;35(32):10493-505 PMID: 8756706
  46. Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups.
    Biochemistry. 1991 Nov 5;30(44):10632-40 PMID: 1931984
  47. A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate.
    Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):542-7 PMID: 9435228
  48. Metal ion catalysis during splicing of premessenger RNA.
    Nature. 1997 Aug 21;388(6644):801-5 PMID: 9285595
  49. Metal-binding sites in the major groove of a large ribozyme domain.
    Structure. 1996 Oct 15;4(10):1221-9 PMID: 8939748
  50. Exocyclic amine of the conserved G.U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5'-splice site selection and transition state stabilization.
    Biochemistry. 1996 Jan 30;35(4):1201-11 PMID: 8573575
  51. Manganese Enzymes with Binuclear Active Sites.
    Chem Rev. 1996 Nov 7;96(7):2909-2926 PMID: 11848845
  52. A second catalytic metal ion in group I ribozyme.
    Nature. 1997 Aug 21;388(6644):805-8 PMID: 9285596
  53. Structural studies of metal binding by inositol monophosphatase: evidence for two-metal ion catalysis.
    Biochemistry. 1994 Aug 16;33(32):9468-76 PMID: 8068621
  54. Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA.
    Proc Natl Acad Sci U S A. 1978 Jan;75(1):64-8 PMID: 343112
  55. Visualizing the higher order folding of a catalytic RNA molecule.
    Science. 1991 Jan 25;251(4992):401-7 PMID: 1989074
  56. Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P.
    Biochemistry. 1995 Jan 24;34(3):902-9 PMID: 7827048
  57. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme.
    Biochemistry. 1992 Feb 11;31(5):1386-99 PMID: 1736996
  58. Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A resolution.
    Biochemistry. 1994 Aug 9;33(31):9333-42 PMID: 8049235
  59. Metal ion interaction with cosubstrate in self-splicing of group I introns.
    Nucleic Acids Res. 1997 Feb 1;25(3):648-53 PMID: 9016608
  60. Isolation of a local tertiary folding transition in the context of a globally folded RNA.
    Nat Struct Biol. 1996 Aug;3(8):701-10 PMID: 8756329
  61. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site.
    Biochemistry. 1990 Nov 6;29(44):10172-80 PMID: 2271646
  62. Determination of the rate of hexokinase-glucose dissociation by the isotope-trapping method.
    J Biol Chem. 1974 Aug 25;249(16):5163-8 PMID: 4604308
  63. Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?
    Biochemistry. 1997 Sep 23;36(38):11389-401 PMID: 9298958
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.96.22.12299
Published
1999-10-26
Pages
12299-304
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC22911
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.96.22.12299 PMC Full Text View journal details
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