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

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Laboratoire de Synthèse de Biomolécules, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Université Claude Bernard Lyon 1.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2011. Vol. 108, no 1, 79-84 p.
Keyword [en]
Chromogranins, Granin-derived peptides, Granins, Immunohistochemistry, Neuroendocrine differentiation, Neuroendocrine tumours, Prohormone convertases, Secretogranins
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-145815DOI: 10.1073/pnas.1012612107ISI: 000285915000019PubMedID: 21169502OAI: oai:DiVA.org:uu-145815DiVA: diva2:396916
Available from: 2011-02-11 Created: 2011-02-11 Last updated: 2017-12-11Bibliographically 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
2. Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
Open this publication in new window or tab >>Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids?

Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition.

By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches.

We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis.

Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1203
Keyword
Ribosome, protein synthesis, translation, efficiency-accuracy trade-off, kinetics, unnatural amino acids
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-235534 (URN)978-91-554-9103-1 (ISBN)
Public defence
2014-12-17, B41, Uppsala biomedical center (BMC), Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2014-11-25 Created: 2014-11-05 Last updated: 2015-02-03

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Authority records BETA

Johansson, Magnus

Search in DiVA

By author/editor
Johansson, Magnus
By organisation
Structure and Molecular Biology
In the same journal
Proceedings of the National Academy of Sciences of the United States of America
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 428 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf