This grant focuses on the catalytic reaction mechanisms of the ribosome and the group I self-splicing intron. Both of these large RNA enzymes (ribozymes) perform biologically essential, but mechanistically distinct chemical reactions. Recent crystallographic evidence demonstrates that the peptidyl transferase center of the 50S ribosomal subunit is comprised entirely of RNA. This suggests that the ribosome is a ribozyme. Based upon the high conservation of the active site nucleotides, an RNA active site is likely to be the catalyst of protein synthesis within all living organisms. Crystallographic and biochemical data support a 23S rRNA catalyzed general acid-base mechanism, wherein nucleotide A2451 (E. coli numbering) transfers a proton from the nucleophilic amine (general base catalysis) to the leaving group hydroxyl (general acid catalysis). This is analogous to the role played by the active site histidine within serine proteases. A2451 can play this role in peptide bond formation because it has a neutral PKa, which represents an increase of at least 4 pH units from the unperturbed value of adenosine. This proposal aims to test the general acid-base mechanism of protein synthesis, to define which position of the A2451 nucleobase has a perturbed PKa, and to determine how the microenvironment of the ribosomal active site causes such a substantial PKa perturbation. Peptide bond formation is one of the most fundamental biochemical reactions and the target of several classes of antibiotics. This proposal aims to define how RNA catalyzes this reaction. The group I intron is a metalloenzyme that catalyzes two consecutive transesterification reactions in the course of RNA self-splicing. In these reactions, the ribozyme binds two substrates, the 5'-exon and the exogenous guanosine. It also positions at least three catalytic metal ions within its active site. This proposal will utilize nucleotide analog approaches to define the binding orientation of the guanosine within the intron active site and to define the active site ligands for the catalytic metal ions. This will lead to improved mechanistic understanding of how RNA can catalyze reactions involved in RNA processing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Lewis, Catherine D
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Yale University
Schools of Medicine
New Haven
United States
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Dunican, Brian F; Hiller, David A; Strobel, Scott A (2015) Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE. Biochemistry 54:7048-57
Smith, Kathryn D; Gordon, Patricia B; Rivetta, Alberto et al. (2015) Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains. J Biol Chem 290:19874-87
Griffin, Meghan A; Davis, Jared H; Strobel, Scott A (2013) Bacterial toxin RelE: a highly efficient ribonuclease with exquisite substrate specificity using atypical catalytic residues. Biochemistry 52:8633-42
Carrasco, Nicolas; Hiller, David A; Strobel, Scott A (2011) Minimal transition state charge stabilization of the oxyanion during peptide bond formation by the ribosome. Biochemistry 50:10491-8
Davis, Jared H; Dunican, Brian F; Strobel, Scott A (2011) glmS Riboswitch binding to the glucosamine-6-phosphate ?-anomer shifts the pKa toward neutrality. Biochemistry 50:7236-42
Hiller, David A; Singh, Vipender; Zhong, Minghong et al. (2011) A two-step chemical mechanism for ribosome-catalysed peptide bond formation. Nature 476:236-9
Hiller, David A; Zhong, Minghong; Singh, Vipender et al. (2010) Transition states of uncatalyzed hydrolysis and aminolysis reactions of a ribosomal P-site substrate determined by kinetic isotope effects. Biochemistry 49:3868-78
Kingery, David A; Pfund, Emmanuel; Voorhees, Rebecca M et al. (2008) An uncharged amine in the transition state of the ribosomal peptidyl transfer reaction. Chem Biol 15:493-500
Zhong, Minghong; Strobel, Scott A (2008) Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction. J Org Chem 73:603-11
Huang, Kevin S; Carrasco, Nicolas; Pfund, Emmanuel et al. (2008) Transition state chirality and role of the vicinal hydroxyl in the ribosomal peptidyl transferase reaction. Biochemistry 47:8822-7

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