Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Nov;4(11):1332–1346. doi: 10.1017/s1355838298980979

Structure-function relationships in the hammerhead ribozyme probed by base rescue.

A Peracchi 1, J Matulic-Adamic 1, S Wang 1, L Beigelman 1, D Herschlag 1
PMCID: PMC1369707  PMID: 9814755

Abstract

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

Full Text

The Full Text of this article is available as a PDF (614.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abramovitz D. L., Friedman R. A., Pyle A. M. Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science. 1996 Mar 8;271(5254):1410–1413. doi: 10.1126/science.271.5254.1410. [DOI] [PubMed] [Google Scholar]
  2. Agris P. F. The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog Nucleic Acid Res Mol Biol. 1996;53:79–129. doi: 10.1016/s0079-6603(08)60143-9. [DOI] [PubMed] [Google Scholar]
  3. Baidya N., Ammons G. E., Matulic-Adamic J., Karpeisky A. M., Beigelman L., Uhlenbeck O. C. Functional groups on the cleavage site pyrimidine nucleotide are required for stabilization of the hammerhead transition state. RNA. 1997 Oct;3(10):1135–1142. [PMC free article] [PubMed] [Google Scholar]
  4. Barrick D., Ho N. T., Simplaceanu V., Dahlquist F. W., Ho C. A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin. Nat Struct Biol. 1997 Jan;4(1):78–83. doi: 10.1038/nsb0197-78. [DOI] [PubMed] [Google Scholar]
  5. Barrick D. Replacement of the proximal ligand of sperm whale myoglobin with free imidazole in the mutant His-93-->Gly. Biochemistry. 1994 May 31;33(21):6546–6554. doi: 10.1021/bi00187a023. [DOI] [PubMed] [Google Scholar]
  6. Bevers S., Xiang G., McLaughlin L. W. Importance of specific adenosine N3-nitrogens for efficient cleavage by a hammerhead ribozyme. Biochemistry. 1996 May 21;35(20):6483–6490. doi: 10.1021/bi952868l. [DOI] [PubMed] [Google Scholar]
  7. Birikh K. R., Heaton P. A., Eckstein F. The structure, function and application of the hammerhead ribozyme. Eur J Biochem. 1997 Apr 1;245(1):1–16. doi: 10.1111/j.1432-1033.1997.t01-3-00001.x. [DOI] [PubMed] [Google Scholar]
  8. Boehlein S. K., Walworth E. S., Richards N. G., Schuster S. M. Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B. J Biol Chem. 1997 May 9;272(19):12384–12392. doi: 10.1074/jbc.272.19.12384. [DOI] [PubMed] [Google Scholar]
  9. Bratty J., Chartrand P., Ferbeyre G., Cedergren R. The hammerhead RNA domain, a model ribozyme. Biochim Biophys Acta. 1993 Dec 14;1216(3):345–359. doi: 10.1016/0167-4781(93)90001-t. [DOI] [PubMed] [Google Scholar]
  10. Carlow D. C., Smith A. A., Yang C. C., Short S. A., Wolfenden R. Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue. Biochemistry. 1995 Apr 4;34(13):4220–4224. doi: 10.1021/bi00013a010. [DOI] [PubMed] [Google Scholar]
  11. Carter P., Abrahmsén L., Wells J. A. Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'. Biochemistry. 1991 Jun 25;30(25):6142–6148. doi: 10.1021/bi00239a009. [DOI] [PubMed] [Google Scholar]
  12. Cerná J. Effect of cytidine-5'-monophosphate on peptidyl transferase activity. FEBS Lett. 1975 Oct 15;58(1):94–98. doi: 10.1016/0014-5793(75)80233-x. [DOI] [PubMed] [Google Scholar]
  13. Chartrand P., Usman N., Cedergren R. Effect of structural modifications on the activity of the leadzyme. Biochemistry. 1997 Mar 18;36(11):3145–3150. doi: 10.1021/bi962219p. [DOI] [PubMed] [Google Scholar]
  14. Dahm S. C., Derrick W. B., Uhlenbeck O. C. Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry. 1993 Dec 7;32(48):13040–13045. doi: 10.1021/bi00211a013. [DOI] [PubMed] [Google Scholar]
  15. Dhalla A. M., Li B., Alibhai M. F., Yost K. J., Hemmingsen J. M., Atkins W. M., Schineller J., Villafranca J. J. Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity. Protein Sci. 1994 Mar;3(3):476–481. doi: 10.1002/pro.5560030313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eriksson A. E., Baase W. A., Wozniak J. A., Matthews B. W. A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature. 1992 Jan 23;355(6358):371–373. doi: 10.1038/355371a0. [DOI] [PubMed] [Google Scholar]
  17. Fitzgerald M. M., Churchill M. J., McRee D. E., Goodin D. B. Small molecule binding to an artificially created cavity at the active site of cytochrome c peroxidase. Biochemistry. 1994 Apr 5;33(13):3807–3818. [PubMed] [Google Scholar]
  18. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  19. Frillingos S., Kaback H. R. Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli. Biochemistry. 1996 Oct 15;35(41):13363–13367. doi: 10.1021/bi961453c. [DOI] [PubMed] [Google Scholar]
  20. Fu D. J., McLaughlin L. W. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry. 1992 Nov 17;31(45):10941–10949. doi: 10.1021/bi00160a001. [DOI] [PubMed] [Google Scholar]
  21. Fu D. J., McLaughlin L. W. Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3985–3989. doi: 10.1073/pnas.89.9.3985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fu D. J., Rajur S. B., McLaughlin L. W. Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme. Biochemistry. 1993 Oct 12;32(40):10629–10637. doi: 10.1021/bi00091a013. [DOI] [PubMed] [Google Scholar]
  23. Hall K. B., Sampson J. R., Uhlenbeck O. C., Redfield A. G. Structure of an unmodified tRNA molecule. Biochemistry. 1989 Jul 11;28(14):5794–5801. doi: 10.1021/bi00440a014. [DOI] [PubMed] [Google Scholar]
  24. Harpel M. R., Hartman F. C. Chemical rescue by exogenous amines of a site-directed mutant of ribulose 1,5-bisphosphate carboxylase/oxygenase that lacks a key lysyl residue. Biochemistry. 1994 May 10;33(18):5553–5561. doi: 10.1021/bi00184a026. [DOI] [PubMed] [Google Scholar]
  25. Herschlag D. RNA chaperones and the RNA folding problem. J Biol Chem. 1995 Sep 8;270(36):20871–20874. doi: 10.1074/jbc.270.36.20871. [DOI] [PubMed] [Google Scholar]
  26. Hertel K. J., Herschlag D., Uhlenbeck O. C. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry. 1994 Mar 22;33(11):3374–3385. doi: 10.1021/bi00177a031. [DOI] [PubMed] [Google Scholar]
  27. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huang S., Tu S. C. Identification and characterization of a catalytic base in bacterial luciferase by chemical rescue of a dark mutant. Biochemistry. 1997 Dec 2;36(48):14609–14615. doi: 10.1021/bi9722554. [DOI] [PubMed] [Google Scholar]
  29. Kim J., Ruzicka F., Frey P. A. Remodeling hexose-1-phosphate uridylyltransferase: mechanism-inspired mutation into a new enzyme, UDP-hexose synthase. Biochemistry. 1990 Nov 27;29(47):10590–10593. doi: 10.1021/bi00499a003. [DOI] [PubMed] [Google Scholar]
  30. Kim S., Liang J., Barry B. A. Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14406–14411. doi: 10.1073/pnas.94.26.14406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kintanar A., Yue D., Horowitz J. Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA. Biochimie. 1994;76(12):1192–1204. doi: 10.1016/0300-9084(94)90049-3. [DOI] [PubMed] [Google Scholar]
  32. Lu Z., Nagata S., McPhie P., Miles E. W. Lysine 87 in the beta subunit of tryptophan synthase that forms an internal aldimine with pyridoxal phosphate serves critical roles in transimination, catalysis, and product release. J Biol Chem. 1993 Apr 25;268(12):8727–8734. [PubMed] [Google Scholar]
  33. Matulic-Adamic Jasenka, Beigelman Leonid, Portmann Stefan, Egli Martin, Usman Nassim. Synthesis and Structure of 1-Deoxy-1-phenyl-beta-D-ribofuranose and Its Incorporation into Oligonucleotides. J Org Chem. 1996 May 31;61(11):3909–3911. doi: 10.1021/jo960091b. [DOI] [PubMed] [Google Scholar]
  34. McKay D. B. Structure and function of the hammerhead ribozyme: an unfinished story. RNA. 1996 May;2(5):395–403. [PMC free article] [PubMed] [Google Scholar]
  35. Morton A., Baase W. A., Matthews B. W. Energetic origins of specificity of ligand binding in an interior nonpolar cavity of T4 lysozyme. Biochemistry. 1995 Jul 11;34(27):8564–8575. doi: 10.1021/bi00027a006. [DOI] [PubMed] [Google Scholar]
  36. Morton A., Matthews B. W. Specificity of ligand binding in a buried nonpolar cavity of T4 lysozyme: linkage of dynamics and structural plasticity. Biochemistry. 1995 Jul 11;34(27):8576–8588. doi: 10.1021/bi00027a007. [DOI] [PubMed] [Google Scholar]
  37. Murray J. B., Adams C. J., Arnold J. R., Stockley P. G. The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site. Biochem J. 1995 Oct 15;311(Pt 2):487–494. doi: 10.1042/bj3110487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murray J. B., Terwey D. P., Maloney L., Karpeisky A., Usman N., Beigelman L., Scott W. G. The structural basis of hammerhead ribozyme self-cleavage. Cell. 1998 Mar 6;92(5):665–673. doi: 10.1016/s0092-8674(00)81134-4. [DOI] [PubMed] [Google Scholar]
  39. Narlikar G. J., Khosla M., Usman N., Herschlag D. 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):2465–2477. doi: 10.1021/bi9610820. [DOI] [PubMed] [Google Scholar]
  40. Newmyer S. L., de Montellano P. R. Rescue of the catalytic activity of an H42A mutant of horseradish peroxidase by exogenous imidazoles. J Biol Chem. 1996 Jun 21;271(25):14891–14896. doi: 10.1074/jbc.271.25.14891. [DOI] [PubMed] [Google Scholar]
  41. Ng M. M., Benseler F., Tuschl T., Eckstein F. Isoguanosine substitution of conserved adenosines in the hammerhead ribozyme. Biochemistry. 1994 Oct 11;33(40):12119–12126. doi: 10.1021/bi00206a015. [DOI] [PubMed] [Google Scholar]
  42. Peracchi A., Beigelman L., Scott E. C., Uhlenbeck O. C., Herschlag D. Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation. J Biol Chem. 1997 Oct 24;272(43):26822–26826. doi: 10.1074/jbc.272.43.26822. [DOI] [PubMed] [Google Scholar]
  43. Peracchi A., Beigelman L., Usman N., Herschlag D. Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11522–11527. doi: 10.1073/pnas.93.21.11522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perona J. J., Hedstrom L., Wagner R. L., Rutter W. J., Craik C. S., Fletterick R. J. Exogenous acetate reconstitutes the enzymatic activity of trypsin Asp189Ser. Biochemistry. 1994 Mar 22;33(11):3252–3259. doi: 10.1021/bi00177a016. [DOI] [PubMed] [Google Scholar]
  45. Perret V., Garcia A., Puglisi J., Grosjean H., Ebel J. P., Florentz C., Giegé R. Conformation in solution of yeast tRNA(Asp) transcripts deprived of modified nucleotides. Biochimie. 1990 Oct;72(10):735–743. doi: 10.1016/0300-9084(90)90158-d. [DOI] [PubMed] [Google Scholar]
  46. Phillips M. A., Hedstrom L., Rutter W. J. Guanidine derivatives restore activity to carboxypeptidase lacking arginine-127. Protein Sci. 1992 Apr;1(4):517–521. doi: 10.1002/pro.5560010406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  48. Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
  49. Rynkiewicz M. J., Seaton B. A. Chemical rescue by guanidine derivatives of an arginine-substituted site-directed mutant of Escherichia coli ornithine transcarbamylase. Biochemistry. 1996 Dec 17;35(50):16174–16179. doi: 10.1021/bi961311i. [DOI] [PubMed] [Google Scholar]
  50. Schmidt S., Beigelman L., Karpeisky A., Usman N., Sorensen U. S., Gait M. J. Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. 1996 Feb 15;24(4):573–581. doi: 10.1093/nar/24.4.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Scott W. G., Finch J. T., Klug A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell. 1995 Jun 30;81(7):991–1002. doi: 10.1016/s0092-8674(05)80004-2. [DOI] [PubMed] [Google Scholar]
  52. Scott W. G., Murray J. B., Arnold J. R., Stoddard B. L., Klug A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science. 1996 Dec 20;274(5295):2065–2069. doi: 10.1126/science.274.5295.2065. [DOI] [PubMed] [Google Scholar]
  53. Seela F., Debelak H., Usman N., Burgin A., Beigelman L. 1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes. Nucleic Acids Res. 1998 Feb 15;26(4):1010–1018. doi: 10.1093/nar/26.4.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sekimoto T., Matsuyama T., Fukui T., Tanizawa K. Evidence for lysine 80 as general base catalyst of leucine dehydrogenase. J Biol Chem. 1993 Dec 25;268(36):27039–27045. [PubMed] [Google Scholar]
  55. Sigler P. B. An analysis of the structure of tRNA. Annu Rev Biophys Bioeng. 1975;4(00):477–527. doi: 10.1146/annurev.bb.04.060175.002401. [DOI] [PubMed] [Google Scholar]
  56. Slim G., Gait M. J. The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage. Biochem Biophys Res Commun. 1992 Mar 16;183(2):605–609. doi: 10.1016/0006-291x(92)90525-p. [DOI] [PubMed] [Google Scholar]
  57. Smith H. B., Hartman F. C. Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue. Biochemistry. 1991 May 28;30(21):5172–5177. doi: 10.1021/bi00235a009. [DOI] [PubMed] [Google Scholar]
  58. Toney M. D., Kirsch J. F. Brønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant. Protein Sci. 1992 Jan;1(1):107–119. doi: 10.1002/pro.5560010111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Toney M. D., Kirsch J. F. Direct Brønsted analysis of the restoration of activity to a mutant enzyme by exogenous amines. Science. 1989 Mar 17;243(4897):1485–1488. doi: 10.1126/science.2538921. [DOI] [PubMed] [Google Scholar]
  60. Ushida C., Muramatsu T., Mizushima H., Ueda T., Watanabe K., Stetter K. O., Crain P. F., McCloskey J. A., Kuchino Y. Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet). Biochimie. 1996;78(10):847–855. doi: 10.1016/s0300-9084(97)84337-4. [DOI] [PubMed] [Google Scholar]
  61. Wincott F., DiRenzo A., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C., Scaringe S., Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 Jul 25;23(14):2677–2684. doi: 10.1093/nar/23.14.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yue D., Kintanar A., Horowitz J. Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation. Biochemistry. 1994 Aug 2;33(30):8905–8911. doi: 10.1021/bi00196a007. [DOI] [PubMed] [Google Scholar]
  63. Zhukovsky E. A., Robinson P. R., Oprian D. D. Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore. Science. 1991 Feb 1;251(4993):558–560. doi: 10.1126/science.1990431. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES