|
1
|
Zhou W, Ding J, Liu J. An Efficient Lanthanide-Dependent DNAzyme Cleaving 2'-5'-Linked RNA. Chembiochem 2016; 17:890-4. [PMID: 26957420 DOI: 10.1002/cbic.201500690] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Indexed: 12/29/2022]
Abstract
RNA can form two types of linkage. In addition to the predominant 3'-5' linkage, 2'-5'-linked RNA is also important in biology, medicine, and prebiotic studies. Here, in vitro selection was used to isolate a DNAzyme that specifically cleaves 2'-5' RNA by using Ce(3+) as the metal cofactor, but leaves the 3'-5' counterpart intact. This Ce5 DNAzyme requires trivalent light lanthanide ions and shows a rate of 0.16 min(-1) in the presence of 10 μm Ce(3+) ; the activity decreases with heavier lanthanide ions. This is the fastest DNAzyme reported for this reaction, and it might enable applications in chemical biology. As a proof-of-concept, using this DNAzyme, the reactions between phosphorothioate-modified RNA and strongly thiophilic metals (Hg(2+) and Tl(3+) ) were studied as a function of pH.
Collapse
Affiliation(s)
- Wenhu Zhou
- School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, 410013, China.,Department of Chemistry and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada
| | - Jinsong Ding
- School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, 410013, China
| | - Juewen Liu
- School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, 410013, China. .,Department of Chemistry and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada.
| |
Collapse
|
|
2
|
Lee TS, Radak BK, Harris ME, York DM. A Two-Metal-Ion-Mediated Conformational Switching Pathway for HDV Ribozyme Activation. ACS Catal 2016; 6:1853-1869. [PMID: 27774349 DOI: 10.1021/acscatal.5b02158] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
RNA enzymes serve as a potentially powerful platform from which to design catalysts and engineer new biotechnology. A fundamental understanding of these systems provides insight to guide design. The hepatitis delta virus ribozyme (HDVr) is a small, self-cleaving RNA motif widely distributed in nature, that has served as a paradigm for understanding basic principles of RNA catalysis. Nevertheless, questions remain regarding the precise roles of divalent metal ions and key nucleotides in catalysis. In an effort to establish a reaction mechanism model consistent with available experimental data, we utilize molecular dynamics simulations to explore different conformations and metal ion binding modes along the HDVr reaction path. Building upon recent crystallographic data, our results provide a dynamic model of the HDVr reaction mechanism involving a conformational switch between multiple non-canonical G25:U20 base pair conformations in the active site. These local nucleobase dynamics play an important role in catalysis by modulating the metal binding environments of two Mg2+ ions that support catalysis at different steps of the reaction pathway. The first ion plays a structural role by inducing a base pair flip necessary to obtain the catalytic fold in which C75 moves towards to the scissile phosphate in the active site. Ejection of this ion then permits a second ion to bind elsewhere in the active site and facilitate nucleophile activation. The simulations collectively describe a mechanistic scenario that is consistent with currently available experimental data from crystallography, phosphorothioate substitutions, and chemical probing studies. Avenues for further experimental verification are suggested.
Collapse
Affiliation(s)
- Tai-Sung Lee
- Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States
| | - Brian K. Radak
- Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States
- Argonne National Laboratory, Argonne, Illinois 60439, United State
| | - Michael E. Harris
- Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States
| | - Darrin M. York
- Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States
| |
Collapse
|
|
3
|
Riccitelli N, Lupták A. HDV family of self-cleaving ribozymes. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2014; 120:123-71. [PMID: 24156943 DOI: 10.1016/b978-0-12-381286-5.00004-4] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The hepatitis delta virus (HDV) ribozymes are catalytic RNAs capable of cleaving their own sugar-phosphate backbone. The HDV virus possesses the ribozymes in both sense and antisense genomic transcripts, where they are essential for processing during replication. These ribozymes have been the subject of intense biochemical scrutiny and have yielded a wealth of mechanistic insights. In recent years, many HDV-like ribozymes have been identified in nearly all branches of life. The ribozymes are implicated in a variety of biological events, including episodic memory in mammals and retrotransposition in many eukaryotes. Detailed analysis of additional HDV-like ribozyme isolates will likely reveal many more biological functions and provide information about the evolution of this unique RNA.
Collapse
Affiliation(s)
- Nathan Riccitelli
- Department of Chemistry, University of California, Irvine, California, USA
| | | |
Collapse
|
|
4
|
Functional RNAs exhibit tolerance for non-heritable 2'-5' versus 3'-5' backbone heterogeneity. Nat Chem 2013; 5:390-4. [PMID: 23609089 PMCID: PMC4088963 DOI: 10.1038/nchem.1623] [Citation(s) in RCA: 81] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2012] [Accepted: 03/12/2013] [Indexed: 11/08/2022]
Abstract
A plausible process for non-enzymatic RNA replication would greatly simplify models of the transition from prebiotic chemistry to simple biology. However, all known conditions for the chemical copying of an RNA template result in the synthesis of a complementary strand that contains a mixture of 2'-5' and 3'-5' linkages, rather than the selective synthesis of only 3'-5' linkages as found in contemporary RNA. Here we show that such backbone heterogeneity is compatible with RNA folding into defined three-dimensional structures that retain molecular recognition and catalytic properties and, therefore, would not prevent the evolution of functional RNAs such as ribozymes. Moreover, the same backbone heterogeneity lowers the melting temperature of RNA duplexes that would otherwise be too stable for thermal strand separation. By allowing copied strands to dissociate, this heterogeneity may have been one of the essential features that allowed RNA to emerge as the first biopolymer.
Collapse
|
|
5
|
Lee TS, Giambaşu G, Harris ME, York DM. Characterization of the Structure and Dynamics of the HDV Ribozyme at Different Stages Along the Reaction Path. J Phys Chem Lett 2011; 2:2538-2543. [PMID: 22200005 PMCID: PMC3244300 DOI: 10.1021/jz201106y] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
The structure and dynamics of the hepatitis delta virus ribozyme (HDVr) are studies using molecular dynamics simulations at several stages along its catalytic reaction path, including reactant, activated precursor, transition state mimic and product states, departing from an initial structure based on the C75U mutant crystal structure (PDB: 1VC7). Results of five 350 ns molecular dynamics simulations reveal a spontaneous rotation of U-1 that leads to an in-line conformation and support the role of protonated C75 as the general acid in the transition state. Our results provide rationale for the interpretation of several important experimental results, and make experimentally testable predictions regarding the roles of key active site residues that are not obvious from any available crystal structures.
Collapse
|
|
6
|
Chen JH, Yajima R, Chadalavada DM, Chase E, Bevilacqua PC, Golden BL. A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage. Biochemistry 2010; 49:6508-18. [DOI: 10.1021/bi100670p] [Citation(s) in RCA: 104] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Affiliation(s)
- Jui-Hui Chen
- Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47906
| | - Rieko Yajima
- Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802
| | - Durga M. Chadalavada
- Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802
| | - Elaine Chase
- Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47906
| | - Philip C. Bevilacqua
- Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802
| | - Barbara L. Golden
- Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47906
| |
Collapse
|
|
7
|
Gong B, Chen JH, Bevilacqua PC, Golden BL, Carey PR. Competition between Co(NH(3)(6)3+ and inner sphere Mg2+ ions in the HDV ribozyme. Biochemistry 2010; 48:11961-70. [PMID: 19888753 DOI: 10.1021/bi901091v] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg(2+) are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH(3))(6)(3+), an analogue of Mg(H(2)O)(6)(2+). Here, the binding of Mg(2+) and Co(NH(3))(6)(3+) to the HDV ribozyme is studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg(2+) alone is introduced to the ribozyme, inner sphere coordination of Mg(H(2)O)(x)(2+) (x </= 5) to nonbridging PO(2)(-) oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg(2+) induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH(3))(6)(3+) alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg(2+) binding, Co(NH(3))(6)(3+) binding does not perturb PO(2)(-) group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH(3))(6)(3+) ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO(2)(-) groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH(3))(6)(3+) ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg(2+)-PO(2)(-) binding sites: sites that Co(NH(3))(6)(3+) can displace and others it cannot.
Collapse
Affiliation(s)
- Bo Gong
- Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA
| | | | | | | | | |
Collapse
|
|
8
|
Feng L, Li F, Zheng X, Pan W, Zhou K, Liu Y, He H, Chen L. The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3' ends in a plasmid-only-based virus rescue system. Arch Virol 2009; 154:1151-6. [PMID: 19526191 DOI: 10.1007/s00705-009-0422-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2008] [Accepted: 06/01/2009] [Indexed: 11/25/2022]
Abstract
Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3' end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.
Collapse
Affiliation(s)
- Liqiang Feng
- Center for Vaccines and Biotherapeutics, Guangzhou Institute of Biomedicine and Health, China
| | | | | | | | | | | | | | | |
Collapse
|
|
9
|
Chen JH, Gong B, Bevilacqua PC, Carey PR, Golden BL. A catalytic metal ion interacts with the cleavage Site G.U wobble in the HDV ribozyme. Biochemistry 2009; 48:1498-507. [PMID: 19178151 DOI: 10.1021/bi8020108] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.
Collapse
Affiliation(s)
- Jui-Hui Chen
- Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907, USA
| | | | | | | | | |
Collapse
|
|
10
|
Cerrone-Szakal AL, Siegfried NA, Bevilacqua PC. Mechanistic characterization of the HDV genomic ribozyme: solvent isotope effects and proton inventories in the absence of divalent metal ions support C75 as the general acid. J Am Chem Soc 2008; 130:14504-20. [PMID: 18842044 DOI: 10.1021/ja801816k] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg(2+) ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg(2+)-independent and two Mg(2+)-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg(2+)-free channel is inverted relative to the fully Mg(2+)-dependent channel, with each having a near-neutral pKa. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg(2+). However, subsequent studies on a double mutant (DM) ribozyme suggested that the pKa observed for WT in the absence of Mg(2+) arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg(2+). Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg(2+)-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Brønsted base appears to be water or hydroxide ion depending on pH. The observed pKa's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg(2+) being similar to that in the presence of Mg(2+) and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Brønsted base.
Collapse
Affiliation(s)
- Andrea L Cerrone-Szakal
- Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
| | | | | |
Collapse
|
|
11
|
Torelli AT, Spitale RC, Krucinska J, Wedekind JE. Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme. Biochem Biophys Res Commun 2008; 371:154-8. [PMID: 18423397 DOI: 10.1016/j.bbrc.2008.04.036] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2008] [Accepted: 04/04/2008] [Indexed: 10/22/2022]
Abstract
Reaction-intermediate analogs have been used to understand how phosphoryl transfer enzymes promote catalysis. Herein we report the first structure of a small ribozyme crystallized with a 3'-OH, 2',5'-linkage in lieu of the normal phosphodiester substrate. The new structure shares features of the reaction coordinate exhibited in prior ribozyme structures including a vanadate complex that mimicked the oxyphosphorane transition state. As such, the structure exhibits reaction-intermediate traits that allow direct comparison of stabilizing interactions to the 3'-OH, 2',5'-linkage contributed by the RNA enzyme and its protein counterpart, ribonuclease. Clear similarities are observed between the respective structures including hydrogen bonds to the non-bridging oxygens of the scissile phosphate. Other commonalities include carefully poised water molecules that may alleviate charge build-up in the transition state and placement of a positive charge near the leaving group. The advantages of 2',5'-linkages to investigate phosphoryl-transfer reactions are discussed, and argue for their expanded use in structural studies.
Collapse
Affiliation(s)
- Andrew T Torelli
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue Box 712, Rochester, New York 14642, USA
| | | | | | | |
Collapse
|
|
12
|
Brown AL, Perrotta AT, Wadkins TS, Been MD. The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme. Nucleic Acids Res 2008; 36:2990-3000. [PMID: 18388129 PMCID: PMC2396440 DOI: 10.1093/nar/gkn156] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022] Open
Abstract
The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1–2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.
Collapse
Affiliation(s)
- Abigail L Brown
- Department of Biochemistry, Duke University Medical Center, Durham NC 27710, USA
| | | | | | | |
Collapse
|
|
13
|
Thayer J, Rao S, Puri N. Detection of Aberrant 2′‐5′ Linkages in RNA by Anion Exchange. ACTA ACUST UNITED AC 2008; Chapter 10:Unit 10.13. [DOI: 10.1002/0471142700.nc1013s32] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
| | | | - Nitin Puri
- Ambion, an Applied Biosystems Business Austin Texas
| |
Collapse
|
|
14
|
Abstract
A discussion of experimental approaches and theoretical difficulties in the identification of ribozymes with novel catalytic functions. New regulatory RNAs with complex structures have recently been discovered, among them the first catalytic riboswitch, a gene-regulatory RNA sequence with catalytic activity. Here we discuss some of the experimental approaches and theoretical difficulties attached to the identification of new ribozymes in genomes.
Collapse
Affiliation(s)
- Christian Hammann
- Research Group Molecular Interactions, Department of Genetics, FB 18 Naturwissenschaften, Universität Kassel, D-34132 Kassel, Germany
| | - Eric Westhof
- Architecture et Réactivité de l'ARN, Université Louis Pasteur de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, CNRS, rue René Descartes, F-67084 Strasbourg Cedex, France
| |
Collapse
|
|
15
|
Torelli AT, Krucinska J, Wedekind JE. A comparison of vanadate to a 2'-5' linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization. RNA (NEW YORK, N.Y.) 2007; 13:1052-70. [PMID: 17488874 PMCID: PMC1894929 DOI: 10.1261/rna.510807] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 02/14/2007] [Accepted: 04/05/2007] [Indexed: 05/15/2023]
Abstract
The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.
Collapse
Affiliation(s)
- Andrew T Torelli
- Department of Biochemistry and Biophysics, Rochester, NY 14642, USA
| | | | | |
Collapse
|
|
16
|
Ke A, Ding F, Batchelor JD, Doudna JA. Structural Roles of Monovalent Cations in the HDV Ribozyme. Structure 2007; 15:281-7. [PMID: 17355864 DOI: 10.1016/j.str.2007.01.017] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2006] [Revised: 12/22/2006] [Accepted: 01/09/2007] [Indexed: 11/22/2022]
Abstract
The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.
Collapse
Affiliation(s)
- Ailong Ke
- Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA
| | | | | | | |
Collapse
|
|
17
|
Thayer JR, Rao S, Puri N, Burnett CA, Young M. Identification of aberrant 2'-5' RNA linkage isomers by pellicular anion exchange chromatography. Anal Biochem 2006; 361:132-9. [PMID: 17161825 DOI: 10.1016/j.ab.2006.10.032] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2006] [Accepted: 10/25/2006] [Indexed: 11/16/2022]
Abstract
During chemical RNA synthesis, many undesired products may be formed. In addition to the "n-x" sequences, depurination products, and incompletely deprotected oligonucleotides, linkage isomers may form during condensation and/or deprotection of the synthetic products. Under acidic conditions, bond migration may alter normal 3'-5' diesters to aberrant 2'-5' diesters. This results in isomers that are difficult to identify by MS and LC-MS techniques because the isomers have identical masses. HPLC methods for identification of these isomers have not advanced because the isomers are not expected to exhibit differences in hydrophobicity that allow resolution by reversed-phase columns. Neither are changes in ionic interactions anticipated for these isomers that would allow resolution by ion exchange methods. We observed that chromatography on pellicular anion exchange phases, but not on porous anion exchange phases, completely resolves oligonucleotides with very slight conformation differences (e.g., DNA vs. RNA of identical sequence). Because incorporation of 2'-5' linkages in RNA will alter solution conformation slightly, we considered that this pellicular ion exchanger might also allow resolution of identical RNA sequences harboring aberrant 2'-5' linkages from those lacking aberrant 2'-5' linkages. Using the nonporous DNAPac PA200 column, we demonstrated a chromatographic procedure for resolving synthetic RNA with aberrant linkages from their normally linked counterparts. Under certain conditions, aberrant isomers are not completely resolved from those containing only normal linkages. Therefore, we also developed an independent linkage-confirming method using a 5'-3' exonuclease. This enzyme produces incomplete digestion products during digestion of synthetic RNA containing aberrant 2'-5' linkages, and these are readily resolved by DNAPac PA200 chromatography.
Collapse
|
|
18
|
Krasovska MV, Sefcikova J, Réblová K, Schneider B, Walter NG, Sponer J. Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme. Biophys J 2006; 91:626-38. [PMID: 16617077 PMCID: PMC1483112 DOI: 10.1529/biophysj.105.079368] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.
Collapse
Affiliation(s)
- Maryna V Krasovska
- Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, Czech Republic
| | | | | | | | | | | |
Collapse
|
|
19
|
Andronescu M, Zhang ZC, Condon A. Secondary structure prediction of interacting RNA molecules. J Mol Biol 2004; 345:987-1001. [PMID: 15644199 DOI: 10.1016/j.jmb.2004.10.082] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2004] [Revised: 10/18/2004] [Accepted: 10/27/2004] [Indexed: 10/26/2022]
Abstract
Computational tools for prediction of the secondary structure of two or more interacting nucleic acid molecules are useful for understanding mechanisms for ribozyme function, determining the affinity of an oligonucleotide primer to its target, and designing good antisense oligonucleotides, novel ribozymes, DNA code words, or nanostructures. Here, we introduce new algorithms for prediction of the minimum free energy pseudoknot-free secondary structure of two or more nucleic acid molecules, and for prediction of alternative low-energy (sub-optimal) secondary structures for two nucleic acid molecules. We provide a comprehensive analysis of our predictions against secondary structures of interacting RNA molecules drawn from the literature. Analysis of our tools on 17 sequences of up to 200 nucleotides that do not form pseudoknots shows that they have 79% accuracy, on average, for the minimum free energy predictions. When the best of 100 sub-optimal foldings is taken, the average accuracy increases to 91%. The accuracy decreases as the sequences increase in length and as the number of pseudoknots and tertiary interactions increases. Our algorithms extend the free energy minimization algorithm of Zuker and Stiegler for secondary structure prediction, and the sub-optimal folding algorithm by Wuchty et al. Implementations of our algorithms are freely available in the package MultiRNAFold.
Collapse
Affiliation(s)
- Mirela Andronescu
- Department of Computer Science, The University of British Columbia, 201-2366 Main Mall, Vancouver, BC, Canada V6T 1Z4.
| | | | | |
Collapse
|
|
20
|
Bevilacqua PC, Brown TS, Nakano SI, Yajima R. Catalytic roles for proton transfer and protonation in ribozymes. Biopolymers 2003; 73:90-109. [PMID: 14691943 DOI: 10.1002/bip.10519] [Citation(s) in RCA: 120] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Utilization of proton transfer in catalysis, which is well known in the mechanisms of protein enzymes, has been described only relatively recently for RNA enzymes. In this article, we present a current understanding of proton transfer by nucleic acids. Rate enhancement and specificity conferred by general acid-base catalysis are discussed. We also present possibilities for electrostatic catalysis from general acids and bases as well as cationic base pairs. The microenvironments of a large RNA provide the possibility of histidine-like pK(a)s for proton transfer, as well as lysine- and arginine-like pK(a)s for electrostatic catalysis. Discussion on proton transfer focuses on the hepatitis delta virus (HDV) and hairpin ribozymes, with select examples drawn from the protein literature. Discussion on electrostatic catalysis also draws on these two ribozymes, and a postulate for electrostatic catalysis by a cationic base pair in the mechanism of peptidyl transfer in the ribosome is presented. We also provide a perspective on possibilities for phosphoryl transfer mechanisms involving phosphorane intermediates and unusual tautomeric forms of the bases. Lastly, a distinction is made between ground state and "transition state" pK(a)s. We favor a model in which changes in pH lead to changes in the distribution of reactive and nonreactive ionizations of the ribozyme molecules in the ground state, and therefore suggest that "pK(a) changes in the transition state" do not provide an acceptable explanation for observed pH-rate profiles.
Collapse
Affiliation(s)
- Philip C Bevilacqua
- Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
| | | | | | | |
Collapse
|
|
21
|
Nishikawa F, Shirai M, Nishikawa S. Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme. EUROPEAN JOURNAL OF BIOCHEMISTRY 2002; 269:5792-803. [PMID: 12444967 DOI: 10.1046/j.1432-1033.2002.03280.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.
Collapse
Affiliation(s)
- Fumiko Nishikawa
- Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan
| | | | | |
Collapse
|
|
22
|
Abstract
The hepatitis delta virus (HDV) ribozymes are self-cleaving RNA sequences critical to the replication of a small RNA genome. A recently determined crystal structure together with biochemical and biophysical studies provides new insight into the possible catalytic mechanism of these ribozymes. The HDV ribozymes are examples of naturally occurring small ribozymes that catalyze cleavage of the RNA backbone with a rate enhancement of 10(6)- to 10(7)-fold over the uncatalyzed rate. To achieve this level of rate enhancement, the HDV ribozymes have been proposed to employ several catalytic strategies that include the use of metal ions, intrinsic binding energy, and a novel example of general acid-base catalysis with a cytosine side chain acting as a proton donor or acceptor.
Collapse
Affiliation(s)
- I-hung Shih
- Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
| | | |
Collapse
|
|
23
|
Abstract
AIM: To explore whether HDV ribozymes have the ability to trans-cleave HCV RNA.
METHODS: Three HDV genomic ribozymes were designed and named RzC1, RzC2 and RzC3. The substrate RNA contained HCV RNA 5’-noncoding region and 5'-fragment of C region (5'-NCR-C). All the ribozymes and HCV RNA 5'-NCR-C were obtained by transcription in vitro from their DNA templates, and HCV RNA 5'-NCR-C was radiolabelled at its 5’-end. Under certain pH, temperature, appropriate concentration of Mg2+ and deionized formamide, these ribozymes were respectively or simultaneously mixed with HCV RNA 5'-NCR-C and reacted for a certain time. The trans-cleavage reaction was stopped at different time points, and the products were separated with polyacrylamide gel electrophoresis (PAGE), displayed by autoradiography. Percentage of trans-cleaved products was measured to indicate the activity of HDV ribozymes.
RESULTS: RzC1 and RzC2 could trans-cleave 26% and 21.8% of HCV RNA 5'-NCR-C under our reaction conditions with 2.5 mol•L-1 deionized formamide respectively. The percentage of HCV RNA 5'-NCR-C trans-cleaved by RzC1, RzC2 or combined usage of the three ribozymes increased with time, up to 24.9%, 20.3% and 37.3% respectively at 90 min point. Almost no product from RzC3 was observed.
CONCLUSION: HDV ribozymes are able to trans-cleave specifically HCV RNA at certain sites under appropriate conditions, and combination of several ribozymes aiming at different target sites can trans-cleave the substrate more efficiently than using only one of them.
Collapse
Affiliation(s)
- Yue-Cheng Yu
- Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing 400038,China.
| | | | | | | | | |
Collapse
|
|
24
|
Wrzesinski J, Legiewicz M, Smólska B, Ciesiolka J. Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions. Nucleic Acids Res 2001; 29:4482-92. [PMID: 11691936 PMCID: PMC60188 DOI: 10.1093/nar/29.21.4482] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3'-terminus. The variants show high cleavage activity in the presence of Mg(2+), Ca(2+) and Mn(2+), lower with Co(2+) and Sr(2+) and some variants are also active with Cd(2+) and Zn(2+) ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg(2+) is partially inhibited in the presence of Na(+), spermidine and some other divalent metal ions. The inert Co(NH(3))(6)(3+) complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.
Collapse
Affiliation(s)
- J Wrzesinski
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
| | | | | | | |
Collapse
|
|
25
|
Lupták A, Ferré-D'Amaré AR, Zhou K, Zilm KW, Doudna JA. Direct pK(a) measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme. J Am Chem Soc 2001; 123:8447-52. [PMID: 11525650 DOI: 10.1021/ja016091x] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used (13)C NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.
Collapse
Affiliation(s)
- A Lupták
- Department of Chemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA
| | | | | | | | | |
Collapse
|
|
26
|
Shih IH, Been MD. Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme. EMBO J 2001; 20:4884-91. [PMID: 11532952 PMCID: PMC125606 DOI: 10.1093/emboj/20.17.4884] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.
Collapse
Affiliation(s)
- I.-hung Shih
- Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Corresponding author e-mail:
| | - Michael D. Been
- Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Corresponding author e-mail:
| |
Collapse
|
|
27
|
Doherty EA, Doudna JA. Ribozyme structures and mechanisms. ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE 2001; 30:457-75. [PMID: 11441810 DOI: 10.1146/annurev.biophys.30.1.457] [Citation(s) in RCA: 133] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
Collapse
Affiliation(s)
- E A Doherty
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
| | | |
Collapse
|
|
28
|
Premraj BJ, Patel PK, Kandimalla ER, Agrawal S, Hosur RV, Yathindra N. NMR structure of a 2',5' RNA favors A type duplex with compact C2'endo nucleotide repeat. Biochem Biophys Res Commun 2001; 283:537-43. [PMID: 11341757 DOI: 10.1006/bbrc.2001.4828] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In order to provide a structural basis for the unusual properties of 2',5' nucleic acids, especially their unsuitability as information molecules, we report here a high resolution NMR structure of a 2',5' RNA fragment r(GCCGCGGC). It forms an A type duplex with C2'endo compact nucleotide repeat, instead of the familiar C3'endo compact nucleotide (seen in RNA) supporting the deductions made earlier from stereochemical considerations. This data together with the observation that 2',5' nucleic acids require mandatory slide and displacement for duplex and triplex structure formation suggest their reluctance to form the biologically relevant B type duplex. It is argued that this lack of flexibility for helical polymorphism and other inadequacies as a consequence of this may be a contributing factor for the rejection of 2',5' links by nature. The structure exhibits interesting features such as the syn glycosyl conformation for the terminal guanine and a hydrogen bond between O3' hydroxyl and anionic oxygen of the phosphate.
Collapse
Affiliation(s)
- B J Premraj
- Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, 600 025, India
| | | | | | | | | | | |
Collapse
|
|
29
|
Abstract
The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
Collapse
Affiliation(s)
- E A Doherty
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
| | | |
Collapse
|
|
30
|
Shih IH, Been MD. Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage. Proc Natl Acad Sci U S A 2001; 98:1489-94. [PMID: 11171978 PMCID: PMC29284 DOI: 10.1073/pnas.98.4.1489] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozymes in D(2)O and H(2)O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K(M) conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.
Collapse
Affiliation(s)
- I H Shih
- Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
| | | |
Collapse
|
|
31
|
Nakano S, Chadalavada DM, Bevilacqua PC. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science 2000; 287:1493-7. [PMID: 10688799 DOI: 10.1126/science.287.5457.1493] [Citation(s) in RCA: 301] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration.
Collapse
Affiliation(s)
- S Nakano
- Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
| | | | | |
Collapse
|