Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.

The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.

[1]  S. Strobel,et al.  Nucleotide analog interference mapping. , 1999, Methods.

[2]  J. Heckman,et al.  Alignment of the two domains of the hairpin ribozyme-substrate complex defined by interdomain photoaffinity crosslinking. , 1999, Journal of molecular biology.

[3]  M. Fedor,et al.  The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects. , 1999, Journal of molecular biology.

[4]  S. Strobel,et al.  A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme. , 1999, Chemistry & biology.

[5]  Frédéric H.-T. Allain,et al.  Solution structure of the loop B domain from the hairpin ribozyme , 1999, Nature Structural Biology.

[6]  M. Boudvillain,et al.  Defining functional groups, core structural features and inter‐domain tertiary contacts essential for group II intron self‐splicing: a NAIM analysis , 1998, The EMBO journal.

[7]  M. Gait,et al.  Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions. , 1998, Nucleic acids research.

[8]  S. Strobel,et al.  A minor groove RNA triple helix within the catalytic core of a group I intron , 1998, Nature Structural Biology.

[9]  N. Walter,et al.  The solvent-protected core of the hairpin ribozyme-substrate complex. , 1998, Biochemistry.

[10]  W. Scott,et al.  The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. , 1998, Chemistry & biology.

[11]  S. Strobel,et al.  Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N2-methylguanosine. , 1998, Biochemistry.

[12]  N. Walter,et al.  Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Lilley,et al.  Folding of the hairpin ribozyme in its natural conformation achieves close physical proximity of the loops. , 1998, Molecular cell.

[14]  R. Gutell,et al.  The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. , 1998, RNA.

[15]  N. Walter,et al.  Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer , 1998, The EMBO journal.

[16]  N. Walter,et al.  The hairpin ribozyme: structure, assembly and catalysis , 1998, Current opinion in chemical biology.

[17]  R. Shippy,et al.  Mutational analysis of loops 1 and 5 of the hairpin ribozyme. , 1998, Biochemistry.

[18]  S. Strobel,et al.  Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site , 1998, Nature Structural Biology.

[19]  E. Westhof,et al.  Inter-domain cross-linking and molecular modelling of the hairpin ribozyme. , 1997, Journal of molecular biology.

[20]  R. Shippy,et al.  Analysis of hairpin ribozyme base mutations in loops 2 and 4 and their effects on cis-cleavage in vitro. , 1997, Biochemistry.

[21]  N. Walter,et al.  Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates. , 1997, RNA.

[22]  S. Strobel,et al.  Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  I. Tinoco,et al.  Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite. , 1996, Biochemistry.

[24]  U. Sørensen,et al.  Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. , 1996, Nucleic acids research.

[25]  M. Fedor,et al.  Kinetics and thermodynamics of intermolecular catalysis by hairpin ribozymes. , 1995, Biochemistry.

[26]  R. Sousa,et al.  A mutant T7 RNA polymerase as a DNA polymerase. , 1995, The EMBO journal.

[27]  G. Krupp,et al.  Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. , 1995, Nucleic acids research.

[28]  J. Grasby,et al.  Purine functional groups in essential residues of the hairpin ribozyme required for catalytic cleavage of RNA. , 1995, Biochemistry.

[29]  S. Butcher,et al.  Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex. , 1994, Journal of molecular biology.

[30]  Y. Komatsu,et al.  Loop-Size Variation To Probe a Bent Structure of a Hairpin Ribozyme , 1994 .

[31]  A Hampel,et al.  Mutagenesis of the hairpin ribozyme. , 1994, Nucleic acids research.

[32]  S. Butcher,et al.  A photo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme. , 1994, Biochemistry.

[33]  J. Grasby,et al.  Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis. , 1994, Biochimie.

[34]  C. Pleij,et al.  Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin. , 1994, Biochimie.

[35]  John M. Burke,et al.  Four ribose 2'-hydroxyl groups essential for catalytic function of the hairpin ribozyme. , 1993, The Journal of biological chemistry.

[36]  S. Butcher,et al.  Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. , 1993, The EMBO journal.

[37]  G. Bruening,et al.  Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA. , 1993, Nucleic acids research.

[38]  Rajesh K. Gaur,et al.  Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme , 1993, Nucleic Acids Res..

[39]  M. Yarus,et al.  Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. , 1992, Journal of molecular biology.

[40]  John M. Burke,et al.  Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes. , 1992, Nucleic acids research.

[41]  John M. Burke,et al.  In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. , 1992, Genes & development.

[42]  G. Steger,et al.  Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus. , 1990, The Journal of general virology.

[43]  R. Tritz,et al.  'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA. , 1990, Nucleic acids research.

[44]  J. M. Buzayan,et al.  Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA. , 1989, Gene.

[45]  W. Gerlach,et al.  Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus. , 1989, Gene.

[46]  R. Tritz,et al.  RNA catalytic properties of the minimum (-)sTRSV sequence. , 1989, Biochemistry.

[47]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[48]  Wolfram Saenger,et al.  Principles of Nucleic Acid Structure , 1983 .

[49]  Y. Kyōgoku,et al.  A linear relationship between electronegativity of 2′-substituents and conformation of adenine nucleosides , 1979 .