Catalytic Mechanism of Peptide Bond Formation on the Ribosome

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The catalytic mechanism of the peptidyltransferase (PT) reaction on the ribosome will be studied. Recent results from ribosome crystallography and from chemical modification of ribosomal RNA (rRNA) suggest that general acid-base catalysis contributes to the PT reaction and that certain bases of rRNA are directly involved in catalysis. However, quantitative data and the direct identification of catalytic groups are lacking so far. The present proposal aims at investigating the kinetic mechanism of the PT reaction by a newly developed quench-flow method, using peptidyl-tRNA as donor and puromycin as acceptor substrate. The contribution of general acid-base catalysis to the PT reaction will be determined by measuring the pH dependence of the reaction rate. By using a puromycin derivative with a hydroxyl group in place of the amino group, the influence of protonating a catalytic group of the ribosome will be distinguished from the protonation of the nucleophile. The presumed catalytic role of certain rRNA bases (A2451, A2447) will be studied kinetically using ribosomes containing mutant 23S rRNA. Finally, the influence of both amino acid composition and chain length of peptidyl-tRNA on the rate of the PT reaction will be studied.