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Choice of tRNA on Translating Ribosomes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Valet av tRNA på translaterande ribosomer (Swedish)
Abstract [en]

This thesis addresses different aspects of the question about accuracy of protein synthesis: i) the mechanism of tRNA selection during translation ii) study of ribosomal mutations that affect accuracy and iii) the choice of aminoacyl-tRNA isoacceptors on synonymous codons.

By measuring the codon reading efficiencies of cognate and near-cognate ternary complexes we demonstrate that in optimal physiological conditions accuracy of substrate selection is much higher than previously reported; that during translation the ribosomal A site is not blocked by unspecific binding of the non-cognate tRNAs which would inhibit the speed of protein synthesis. Our results suggest that there is an asymmetry between initial selection and proofreading step concerning the wobble position, and that binding of non-cognate substrate does not induce GTP hydrolysis on the ribosome.

The knowledge obtained from the ribosomal mutant strains can be used to explain the general relation between the structure of the ribosome and the mechanism of codon recognition, as well as the streptomycin resistance or dependence phenomenon.

Our work showed experimentally that the probability for binding certain tRNA to the A site of the ribosome is not based on the simple codon-anticodon base pair matching. In the living cell the availability of cognate tRNAs versus the demand for them (the frequency of codon usage) is finely balanced to ensure critical protein synthesis in stress conditions. We have also discovered a new codon assignment for a specific tRNALeu isoacceptor and detected a base modification in its anticodon, which has not been previously observed. The motivation for the later findings comes from a system biology modeling and the results are an example of an interdisciplinary collaboration.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2006. , p. 39
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 143
Keywords [en]
Molecular biology, Ribosome, Translation, Accuracy, Proofreading, tRNA
Keywords [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-6324ISBN: 91-554-6454-8 (print)OAI: oai:DiVA.org:uu-6324DiVA, id: diva2:167662
Public defence
2006-02-17, Room B22, BMC, Husargatan 3, Uppsala, 10:30
Opponent
Supervisors
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2013-03-21Bibliographically approved
List of papers
1. Accuracy of codon recognition in bacterial protein synthesis
Open this publication in new window or tab >>Accuracy of codon recognition in bacterial protein synthesis
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-93985 (URN)
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2010-01-13Bibliographically approved
2. Kinetic properties of unusual ribosomal mutations affecting accuracy of translation
Open this publication in new window or tab >>Kinetic properties of unusual ribosomal mutations affecting accuracy of translation
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-93986 (URN)
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2010-01-13Bibliographically approved
3. Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading
Open this publication in new window or tab >>Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading
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2005 In: Journal of Molecular Biology, Vol. 354, no 1, p. 16-24Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-93987 (URN)
Available from: 2006-01-27 Created: 2006-01-27Bibliographically approved
4. The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy
Open this publication in new window or tab >>The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 7, p. 4548-4556Article in journal (Refereed) Published
Abstract [en]

It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide release by, in particular, the class I release factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (ΔL11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4-6-fold reductions in termination efficiency (kcat/Km) of RF1, but not of RF2, on ΔL11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93990 (URN)10.1074/jbc.M510433200 (DOI)16371360 (PubMedID)
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2017-12-14Bibliographically approved
5. Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA
Open this publication in new window or tab >>Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA
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2005 In: Nucleic Acid Research, Vol. 33, no 11, p. 3529-3539Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-93989 (URN)
Available from: 2006-01-27 Created: 2006-01-27Bibliographically approved

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