The Novel Chemical Mechanism of the Twister Ribozyme.

We describe the multifactorial origins of catalysis by the twister ribozyme. We provide evidence that the adenine immediately 3' to the scissile phosphate (A1) acts as a general acid. Substitution of ring nitrogen atoms indicates that very unusually the N3 of A1 is the proton donor to the oxyanion leaving group. A1 is accommodated in a specific binding pocket that raises its pKa toward neutrality, juxtaposes its N3 with the O5' to be protonated, and helps create the in-line trajectory required for nucleophilic attack. A1 performs general acid catalysis while G33 acts as a general base. A 100-fold stereospecific phosphorothioate effect at the scissile phosphate is consistent with a significant stabilization of the transition state by the ribozyme, and functional group substitution at G33 indicates that its exocyclic N2 interacts directly with the scissile phosphate. A model of the ribozyme active site is proposed that accommodates these catalytic strategies.

[1]  Darrin M. York,et al.  Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation. , 2016, Journal of the American Chemical Society.

[2]  R. Micura,et al.  A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class. , 2015, Angewandte Chemie.

[3]  Phoebe A Rice,et al.  Crystal Structure of the VS ribozyme , 2015, Nature chemical biology.

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

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

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

[7]  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.

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

[9]  J. Piccirilli,et al.  RNA catalyzes nuclear pre-mRNA splicing , 2013, Nature.

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

[11]  H. Sigel,et al.  Understanding the acid-base properties of adenosine: the intrinsic basicities of N1, N3 and N7. , 2011, Chemistry.

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

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

[14]  J. Wedekind,et al.  Identification of an imino group indispensable for cleavage by a small ribozyme. , 2009, Journal of the American Chemical Society.

[15]  J. Wedekind,et al.  Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme. , 2008, RNA.

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

[17]  D. Lilley,et al.  Nucleobase catalysis in the hairpin ribozyme. , 2006, RNA.

[18]  Adam Roth,et al.  Ribozyme speed limits. , 2003, RNA.

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

[20]  A. Ferré-D’Amaré,et al.  Transition State Stabilization by a Catalytic RNA , 2002, Science.

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

[22]  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.

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

[24]  I. Shih,et al.  Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Feigon,et al.  Adenine protonation in domain B of the hairpin ribozyme. , 2000, Biochemistry.

[26]  T. Steitz,et al.  The structural basis of ribosome activity in peptide bond synthesis. , 2000, Science.

[27]  R R Breaker,et al.  Relationship between internucleotide linkage geometry and the stability of RNA. , 1999, RNA.

[28]  F. Seela,et al.  1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes. , 1998, Nucleic acids research.

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

[30]  O. Uhlenbeck,et al.  Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. , 1987, Nucleic acids research.

[31]  G. Cristalli,et al.  Improved synthesis and antitumor activity of 1-deazaadenosine. , 1987, Journal of medicinal chemistry.

[32]  G. Cristalli,et al.  Deaza analogues of adenosine as inhibitors of blood platelet aggregation. , 1984, Journal of pharmaceutical sciences.

[33]  B. Potter,et al.  Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. , 1983, Biochemistry.

[34]  G. Hakimelahi,et al.  High yield selective 3′-silylation of ribonucleosides , 1982 .

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

[36]  S. Benkovic,et al.  Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom. , 1979, Biochemistry.

[37]  Y. Mizuno,et al.  Synthetic studies of potential antimetabolites. XIII. Synthesis of 7-amino-3-β-d-ribofuranosyl-3H-imidazo[4,5-b] pyridine (1-deazaadenosine) and related nucleosides† , 1972 .

[38]  Gerhard Steger,et al.  From alpaca to zebrafish: hammerhead ribozymes wherever you look. , 2011, RNA.

[39]  J. Piccirilli,et al.  Separation of RNA phosphorothioate oligonucleotides by HPLC. , 2009, Methods in enzymology.

[40]  S. Beaucage,et al.  3H-1,2-Benzodithiole-3-one 1,1-dioxide as an improved sulfurizing reagent in the solid-phase synthesis of oligodeoxyribonucleoside phosphorothioates , 1990 .