Abstract
Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.
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- Ban N., Nissen P., Hansen J., Moore P. B., Steitz T. A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905–920. doi: 10.1126/science.289.5481.905. [DOI] [PubMed] [Google Scholar]
- Basu S., Strobel S. A. Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain. RNA. 1999 Nov;5(11):1399–1407. doi: 10.1017/s135583829999115x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Been M. D., Wickham G. S. Self-cleaving ribozymes of hepatitis delta virus RNA. Eur J Biochem. 1997 Aug 1;247(3):741–753. doi: 10.1111/j.1432-1033.1997.00741.x. [DOI] [PubMed] [Google Scholar]
- Ciesiołka J., Michałowski D., Wrzesinski J., Krajewski J., Krzyzosiak W. J. Patterns of cleavages induced by lead ions in defined RNA secondary structure motifs. J Mol Biol. 1998 Jan 16;275(2):211–220. doi: 10.1006/jmbi.1997.1462. [DOI] [PubMed] [Google Scholar]
- Clouet-d'Orval B., Uhlenbeck O. C. Hammerhead ribozymes with a faster cleavage rate. Biochemistry. 1997 Jul 29;36(30):9087–9092. doi: 10.1021/bi9710941. [DOI] [PubMed] [Google Scholar]
- Connell G. J., Yarus M. RNAs with dual specificity and dual RNAs with similar specificity. Science. 1994 May 20;264(5162):1137–1141. doi: 10.1126/science.7513905. [DOI] [PubMed] [Google Scholar]
- Cowan J. A. Metal Activation of Enzymes in Nucleic Acid Biochemistry. Chem Rev. 1998 May 7;98(3):1067–1088. doi: 10.1021/cr960436q. [DOI] [PubMed] [Google Scholar]
- 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]
- Dahm S. C., Uhlenbeck O. C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. doi: 10.1021/bi00103a011. [DOI] [PubMed] [Google Scholar]
- Derrick W. B., Greef C. H., Caruthers M. H., Uhlenbeck O. C. Hammerhead cleavage of the phosphorodithioate linkage. Biochemistry. 2000 Apr 25;39(16):4947–4954. doi: 10.1021/bi000146a. [DOI] [PubMed] [Google Scholar]
- Feig A. L., Panek M., Horrocks W. D., Jr, Uhlenbeck O. C. Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy. Chem Biol. 1999 Nov;6(11):801–810. doi: 10.1016/s1074-5521(99)80127-6. [DOI] [PubMed] [Google Scholar]
- Feig A. L., Scott W. G., Uhlenbeck O. C. Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb. Science. 1998 Jan 2;279(5347):81–84. doi: 10.1126/science.279.5347.81. [DOI] [PubMed] [Google Scholar]
- Ferré-D'Amaré A. R., Zhou K., Doudna J. A. Crystal structure of a hepatitis delta virus ribozyme. Nature. 1998 Oct 8;395(6702):567–574. doi: 10.1038/26912. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Gluick T. C., Wills N. M., Gesteland R. F., Draper D. E. Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability. Biochemistry. 1997 Dec 23;36(51):16173–16186. doi: 10.1021/bi971362v. [DOI] [PubMed] [Google Scholar]
- Hampel A., Cowan J. A. A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage. Chem Biol. 1997 Jul;4(7):513–517. doi: 10.1016/s1074-5521(97)90323-9. [DOI] [PubMed] [Google Scholar]
- Heerschap A., Walters J. A., Hilbers C. W. Interactions of some naturally occurring cations with phenylalanine and initiator tRNA from yeast as reflected by their thermal stability. Biophys Chem. 1985 Aug;22(3):205–217. doi: 10.1016/0301-4622(85)80044-2. [DOI] [PubMed] [Google Scholar]
- Hendler S., Fürer E., Srinivasan P. R. Synthesis and chemical properties of monomers and polymers containing 7-methylguanine and an investigation of their substrate or template properties for bacterial deoxyribonucleic acid or ribonucleic acid polymerases. Biochemistry. 1970 Oct 13;9(21):4141–4153. doi: 10.1021/bi00823a017. [DOI] [PubMed] [Google Scholar]
- Horton T. E., DeRose V. J. Cobalt hexammine inhibition of the hammerhead ribozyme. Biochemistry. 2000 Sep 19;39(37):11408–11416. doi: 10.1021/bi001141g. [DOI] [PubMed] [Google Scholar]
- Hunsicker L. M., DeRose V. J. Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes. J Inorg Biochem. 2000 Jul 1;80(3-4):271–281. doi: 10.1016/s0162-0134(00)00079-9. [DOI] [PubMed] [Google Scholar]
- Labuda D., Augustyniak J. Dependence of tRNA structure in solution upon ionic condition of the solvent. Fluorescence studies of monovalent cation binding to tRNAPhe from barley embryos. Eur J Biochem. 1977 Sep 15;79(1):303–307. doi: 10.1111/j.1432-1033.1977.tb11810.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Murray J. B., Seyhan A. A., Walter N. G., Burke J. M., Scott W. G. The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol. 1998 Oct;5(10):587–595. doi: 10.1016/s1074-5521(98)90116-8. [DOI] [PubMed] [Google Scholar]
- Murray J. B., Seyhan A. A., Walter N. G., Burke J. M., Scott W. G. The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol. 1998 Oct;5(10):587–595. doi: 10.1016/s1074-5521(98)90116-8. [DOI] [PubMed] [Google Scholar]
- Murray J. B., Szöke H., Szöke A., Scott W. G. Capture and visualization of a catalytic RNA enzyme-product complex using crystal lattice trapping and X-ray holographic reconstruction. Mol Cell. 2000 Feb;5(2):279–287. doi: 10.1016/s1097-2765(00)80423-2. [DOI] [PubMed] [Google Scholar]
- Muth G. W., Ortoleva-Donnelly L., Strobel S. A. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center. Science. 2000 Aug 11;289(5481):947–950. doi: 10.1126/science.289.5481.947. [DOI] [PubMed] [Google Scholar]
- Nakano S., Chadalavada D. M., Bevilacqua P. C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science. 2000 Feb 25;287(5457):1493–1497. doi: 10.1126/science.287.5457.1493. [DOI] [PubMed] [Google Scholar]
- Narlikar G. J., Herschlag D. Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. Annu Rev Biochem. 1997;66:19–59. doi: 10.1146/annurev.biochem.66.1.19. [DOI] [PubMed] [Google Scholar]
- Nesbitt S., Hegg L. A., Fedor M. J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem Biol. 1997 Aug;4(8):619–630. doi: 10.1016/s1074-5521(97)90247-7. [DOI] [PubMed] [Google Scholar]
- Nissen P., Hansen J., Ban N., Moore P. B., Steitz T. A. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000 Aug 11;289(5481):920–930. doi: 10.1126/science.289.5481.920. [DOI] [PubMed] [Google Scholar]
- 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]
- Perrotta A. T., Shih I., Been M. D. Imidazole rescue of a cytosine mutation in a self-cleaving ribozyme. Science. 1999 Oct 1;286(5437):123–126. doi: 10.1126/science.286.5437.123. [DOI] [PubMed] [Google Scholar]
- Scott E. C., Uhlenbeck O. C. A re-investigation of the thio effect at the hammerhead cleavage site. Nucleic Acids Res. 1999 Jan 15;27(2):479–484. doi: 10.1093/nar/27.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seyhan A. A., Burke J. M. Mg2+-independent hairpin ribozyme catalysis in hydrated RNA films. RNA. 2000 Feb;6(2):189–198. doi: 10.1017/s1355838200991441. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soukup G. A., Breaker R. R. Relationship between internucleotide linkage geometry and the stability of RNA. RNA. 1999 Oct;5(10):1308–1325. doi: 10.1017/s1355838299990891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stage-Zimmermann T. K., Uhlenbeck O. C. Hammerhead ribozyme kinetics. RNA. 1998 Aug;4(8):875–889. doi: 10.1017/s1355838298980876. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Suga H., Cowan J. A., Szostak J. W. Unusual metal ion catalysis in an acyl-transferase ribozyme. Biochemistry. 1998 Jul 14;37(28):10118–10125. doi: 10.1021/bi980432a. [DOI] [PubMed] [Google Scholar]
- Torrent C., Bordet T., Darlix J. L. Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus. J Mol Biol. 1994 Jul 29;240(5):434–444. doi: 10.1006/jmbi.1994.1459. [DOI] [PubMed] [Google Scholar]
- Urbanke C., Römer R., Maass G. Tertiary structure of tRNAPhe (yeast): kinetics and electrostatic repulsion. Eur J Biochem. 1975 Jul 1;55(2):439–444. doi: 10.1111/j.1432-1033.1975.tb02180.x. [DOI] [PubMed] [Google Scholar]
- Wang S., Karbstein K., Peracchi A., Beigelman L., Herschlag D. Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate. Biochemistry. 1999 Oct 26;38(43):14363–14378. doi: 10.1021/bi9913202. [DOI] [PubMed] [Google Scholar]
- Young K. J., Gill F., Grasby J. A. Metal ions play a passive role in the hairpin ribozyme catalysed reaction. Nucleic Acids Res. 1997 Oct 1;25(19):3760–3766. doi: 10.1093/nar/25.19.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]