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Rate and Accuracy of Bacterial Protein Synthesis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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 [en]
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: urn:nbn:se:uu:diva-171040ISBN: 978-91-554-8309-8 (print)OAI: oai:DiVA.org:uu-171040DiVA: diva2:510129
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
List of papers
1. The kinetics of ribosomal peptidyl transfer revisited
Open this publication in new window or tab >>The kinetics of ribosomal peptidyl transfer revisited
2008 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, 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.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-104034 (URN)10.1016/j.molcel.2008.04.010 (DOI)000256644100009 ()18538657 (PubMedID)
Available from: 2009-05-27 Created: 2009-05-27 Last updated: 2017-12-13Bibliographically approved
2. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
Open this publication in new window or tab >>pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
Show others...
2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 1, 79-84 p.Article in journal (Refereed) Published
Abstract [en]

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

Keyword
Chromogranins, Granin-derived peptides, Granins, Immunohistochemistry, Neuroendocrine differentiation, Neuroendocrine tumours, Prohormone convertases, Secretogranins
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-145815 (URN)10.1073/pnas.1012612107 (DOI)000285915000019 ()21169502 (PubMedID)
Available from: 2011-02-11 Created: 2011-02-11 Last updated: 2017-12-11Bibliographically approved
3. Rate and accuracy of bacterial protein synthesis revisited
Open this publication in new window or tab >>Rate and accuracy of bacterial protein synthesis revisited
2008 (English)In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 11, no 2, 141-147 p.Article, review/survey (Refereed) Published
Abstract [en]

Our understanding of the accuracy of tRNA selection on the messenger RNA programmed ribosome has recently increased dramatically because of high-resolution crystal structures of the ribosome, cryo-electron microscopy reconstructions of its functional complexes, and fast kinetics experiments. Application of single-molecule spectroscopy with fluorescence resonance energy transfer to studies of tRNA selection by the ribosome has also provided new, albeit controversial, insights. Interestingly, when the fundamental trade-off between rate and accuracy in substrate-selective biosynthetic reactions is taken into account, some aspects of the current models of ribosome function appear strikingly suboptimal in the context of growing bacterial cells.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-104032 (URN)10.1016/j.mib.2008.02.015 (DOI)000255806600011 ()18400551 (PubMedID)
Available from: 2009-05-27 Created: 2009-05-27 Last updated: 2017-12-13Bibliographically approved
4. Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
Open this publication in new window or tab >>Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 1, 131-136 p.Article in journal (Refereed) Published
Abstract [en]

Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.

Keyword
fidelity, rate-accuracy trade-off, ribosome, protein synthesis, elongation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-167161 (URN)10.1073/pnas.1116480109 (DOI)000298876500031 ()
Available from: 2012-01-31 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved

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