uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Role of ribosomal protein L27 in peptidyl transfer
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
2008 (English)In: Biochemistry, ISSN 0006-2960, Vol. 47, no 17, 4898-4906 p.Article in journal (Refereed) Published
Abstract [en]

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward pK(a), shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.

Place, publisher, year, edition, pages
2008. Vol. 47, no 17, 4898-4906 p.
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-96711DOI: 10.1021/bi8001874ISI: 000255164700005PubMedID: 18393533OAI: oai:DiVA.org:uu-96711DiVA: diva2:171373
Available from: 2008-02-08 Created: 2008-02-08 Last updated: 2009-11-11Bibliographically 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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Department of Cell and Molecular BiologyStructural Molecular Biology
In the same journal
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 158 hits
ReferencesLink to record
Permanent link

Direct link