The structure of a nucleolytic ribozyme that employs a catalytic metal ion

The TS ribozyme (originally called twister-sister) is a nucleolytic catalytic RNA. We present a crystal structure of the ribozyme in a pre-reactive conformation. Two co-axial helical stacks are organized by a three-way junction and two tertiary contacts. Five divalent metal ions are directly coordinated to RNA ligands, making important contributions to the RNA architecture. The scissile phosphate lies in a quasi-helical loop region that is organized by a network of hydrogen bonding. A divalent metal ion is directly bound to the nucleobase 5' to the scissile phosphate, with an inner-sphere water molecule positioned to interact with the O2' nucleophile. The rate of ribozyme cleavage correlated in a log-linear manner with divalent metal ion pKa, consistent with proton transfer in the transition state, and we propose the bound metal ion is a likely general base for the cleavage reaction. Our data indicate that the TS ribozyme functions predominantly as a metalloenzyme.

[1]  M. Caruthers,et al.  Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis , 1981 .

[2]  High yield selective 3′-silylation of ribonucleosides , 1981 .

[3]  R. Cedergren,et al.  Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity , 1990, Nature.

[4]  N. Seeman,et al.  Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). A nomenclature of junctions and branchpoints in nucleic acids. Recommendations 1994. , 1995, European journal of biochemistry.

[5]  A nomenclature of junctions and branchpoints in nucleic acids. Recommendations 1994: Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) , 1995 .

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

[7]  M. Fedor.  Tertiary structure stabilization promotes hairpin ribozyme ligation. , 1999, Biochemistry.

[8]  D. Lilley,et al.  Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme. , 2001, Biochemistry.

[9]  S. Nakano,et al.  Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism. , 2001, Biochemistry.

[10]  D. Herschlag,et al.  Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. , 2001, Biochemistry.

[11]  F. Major,et al.  Functional involvement of G8 in the hairpin ribozyme cleavage mechanism , 2001, The EMBO journal.

[12]  C. Fierke,et al.  Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P. , 2002, RNA.

[13]  P. Bevilacqua.  Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. , 2003, Biochemistry.

[14]  D. Lilley,et al.  Efficient, pH-dependent RNA ligation by the VS ribozyme in trans. , 2004, Biochemistry.

[15]  R. Breaker,et al.  Control of gene expression by a natural metabolite-responsive ribozyme , 2004, Nature.

[16]  Jennifer A. Doudna,et al.  A conformational switch controls hepatitis delta virus ribozyme catalysis , 2004, Nature.

[17]  D. Lilley,et al.  Observation of internal cleavage and ligation reactions of a ribozyme , 2004, Nature Structural &Molecular Biology.

[18]  M. Fedor,et al.  Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue. , 2004, Journal of molecular biology.

[19]  J. Piccirilli,et al.  General acid catalysis by the hepatitis delta virus ribozyme , 2005, Nature chemical biology.

[20]  John M. Burke,et al.  Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site. , 2005, Biochemistry.

[21]  S. Strobel,et al.  Structural Evidence for a Two-Metal-Ion Mechanism of Group I Intron Splicing , 2005, Science.

[22]  Eric Westhof,et al.  Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thalianaw⃞ , 2005, The Plant Cell Online.

[23]  W. Scott,et al.  Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis , 2006, Cell.

[24]  Andrej Lupták,et al.  A Genomewide Search for Ribozymes Reveals an HDV-Like Sequence in the Human CPEB3 Gene , 2006, Science.

[25]  A. Ferré-D’Amaré,et al.  Structural Basis of glmS Ribozyme Activation by Glucosamine-6-Phosphate , 2006, Science.

[26]  D. Lilley,et al.  A guanine nucleobase important for catalysis by the VS ribozyme , 2007, The EMBO journal.

[27]  S. Strobel,et al.  Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor. , 2007, Chemistry & biology.

[28]  A. Pardi,et al.  Efficient ligation of the Schistosoma hammerhead ribozyme. , 2007, Biochemistry.

[29]  D. Lilley,et al.  Ribozymes and RNA Catalysis , 2007 .

[30]  A. Ferré-D’Amaré,et al.  Essential role of an active-site guanine in glmS ribozyme catalysis. , 2007, Journal of the American Chemical Society.

[31]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[32]  D. Herschlag,et al.  Functional identification of ligands for a catalytic metal ion in group I introns. , 2008, Biochemistry.

[33]  D. York,et al.  Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation. , 2008, Journal of the American Chemical Society.

[34]  Harry F. Noller,et al.  A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA , 2008, Nature.

[35]  A. Pardi,et al.  Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme. , 2008, RNA.

[36]  Andrej Lupták,et al.  Widespread Occurrence of Self-Cleaving Ribozymes , 2009, Science.

[37]  D. Lilley,et al.  The Evolution of Ribozyme Chemistry , 2009, Science.

[38]  Kathryn D. Smith,et al.  Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme. , 2009, Biochemistry.

[39]  D. Perrin,et al.  Probing general acid catalysis in the hammerhead ribozyme. , 2008, Journal of the American Chemical Society.

[40]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[41]  B. Golden,et al.  A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage. , 2010, Biochemistry.

[42]  Tao Pan,et al.  Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA , 2010, Nature.

[43]  D. Lilley,et al.  Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme , 2010, Proceedings of the National Academy of Sciences.

[44]  D. Lilley,et al.  Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid-base catalysis? A critical assessment of available experimental data. , 2011, RNA.

[45]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[46]  D. Herschlag,et al.  Metal-ion rescue revisited: biochemical detection of site-bound metal ions important for RNA folding. , 2012, RNA.

[47]  Jun Lu,et al.  General acid-base catalysis mediated by nucleobases in the hairpin ribozyme. , 2012, Journal of the American Chemical Society.

[48]  B. Golden,et al.  Identification of the catalytic Mg²⁺ ion in the hepatitis delta virus ribozyme. , 2013, Biochemistry.

[49]  Daniel Eiler,et al.  Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme , 2014, Proceedings of the National Academy of Sciences.

[50]  Yijin Liu,et al.  Crystal structure and mechanistic investigation of the twister ribozyme. , 2014, Nature chemical biology.

[51]  R. Breaker,et al.  A widespread self-cleaving ribozyme class is revealed by bioinformatics , 2013, Nature chemical biology.

[52]  E. Westhof,et al.  In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme , 2014, Nature Communications.

[53]  R. Breaker,et al.  New classes of self-cleaving ribozymes revealed by comparative genomics analysis , 2015, Nature chemical biology.

[54]  D. Lilley,et al.  The Novel Chemical Mechanism of the Twister Ribozyme. , 2016, Journal of the American Chemical Society.