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Analysis of Predictions for the Catalytic Mechanism of Ribosomal Peptidyl Transfer
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
2006 In: Biochemistry, ISSN 1520-4995, Vol. 45, no 23, 7049-7056 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2006. Vol. 45, no 23, 7049-7056 p.
URN: urn:nbn:se:uu:diva-96709OAI: oai:DiVA.org:uu-96709DiVA: diva2:171371
Available from: 2008-02-08 Created: 2008-02-08Bibliographically approved
In thesis
1. Computer simulations of ribosome reactions
Open this publication in new window or tab >>Computer simulations of ribosome reactions
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs.

Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination.

New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer. The simulations predict that the protein N terminus interacts with the A-site substrate in a way that promotes binding. The catalytic effect of L27 in the ribosome, however, is shown to be marginal and it therefore seems valid to view the PTC as a ribozyme. Simulations with the model substrate puromycin (Pmn) predicts that protonation of the N terminus can reduce the rate of peptidyl transfer. This could explain the different pH-rate profiles measured for Pmn, compared to other substrates.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 57 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 390
Molecular biology, ribosomal termination, Empirical valence bond, peptidyl transfer, Molekylärbiologi
urn:nbn:se:uu:diva-8429 (URN)978-91-554-7083-8 (ISBN)
Public defence
2008-02-29, B21, BMC, Husarg. 3, Uppsala, 13:00
Available from: 2008-02-08 Created: 2008-02-08Bibliographically approved

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