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The kinetics of ribosomal peptidyl transfer revisited
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. (ehrenberg)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. (ehrenberg)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. (ehrenberg)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. (ehrenberg)
2008 (English)In: Molecular Cell, ISSN 1097-2765, Vol. 30, no 5, 589-598 p.Article in journal (Refereed) Published
Abstract [en]

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

Place, publisher, year, edition, pages
2008. Vol. 30, no 5, 589-598 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-104034DOI: 10.1016/j.molcel.2008.04.010ISI: 000256644100009PubMedID: 18538657OAI: oai:DiVA.org:uu-104034DiVA: diva2:219268
Available from: 2009-05-27 Created: 2009-05-27 Last updated: 2016-04-21Bibliographically approved
In thesis
1. Rate and Accuracy of Bacterial Protein Synthesis
Open this publication in new window or tab >>Rate and Accuracy of Bacterial Protein Synthesis
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates.

Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell.

To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 910
Keyword
protein synthesis, ribosome, peptidyl transfer, rate-accuracy trade-off, kinetics
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-171040 (URN)978-91-554-8309-8 (ISBN)
Public defence
2012-05-04, B41, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2012-04-13 Created: 2012-03-15 Last updated: 2012-04-19

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