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
The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy
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)
Show others and affiliations
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 7, 4548-4556 p.Article 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.

Place, publisher, year, edition, pages
2006. Vol. 281, no 7, 4548-4556 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93990DOI: 10.1074/jbc.M510433200PubMedID: 16371360OAI: oai:DiVA.org:uu-93990DiVA: diva2:167663
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis: Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis
Open this publication in new window or tab >>Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis: Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Mångsidig Användning av ett Förbättrat Cell-Fritt System för Proteinbiosyntes : Funktionella och strukturella studier av ribosomalt protein L11 och klass II release faktor RF3. Ny bioteknologisk metod för kontinuerlig proteinbiosyntes
Abstract [en]

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process.

In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations.

The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site.

In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 94 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 144
Keyword
Molecular biology, Protein synthesis, translation termination, ribosome, release factor, cell-free system, missense error, gel filtration, affinity chromatography, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6325 (URN)91-554-6455-6 (ISBN)
Public defence
2006-02-16, Room B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2012-03-08Bibliographically approved
2. Choice of tRNA on Translating Ribosomes
Open this publication in new window or tab >>Choice of tRNA on Translating Ribosomes
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Valet av tRNA på translaterande ribosomer
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. 39 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 143
Keyword
Molecular biology, Ribosome, Translation, Accuracy, Proofreading, tRNA, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6324 (URN)91-554-6454-8 (ISBN)
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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Molecular Biology
In the same journal
Journal of Biological Chemistry
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 499 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