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Finding the unknowns in trans-translation
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Hitta de okända faktorerna för trans-translation (Swedish)
Abstract [en]

Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called trans-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB).

In this work we have used an in vitro translation system with pure components to further clarify the roles of tmRNA and SmpB in trans-translation.

We found that SmpB binds ribosomes in vivo and in vitro independently of tmRNA presence and is essential for tmRNA binding and trans-peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after trans-peptidation and that SmpB pre-bound to the ribosome can trigger trans-translation.

We demonstrated that the rate of trans-transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that trans-translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action in vivo must always be preceded by mRNA truncation.

We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon.

Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct trans-translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB in vivo and during trans-translation. Specific cleavage patterns of tmRNA were observed at different stages of trans-translation, but the overall tmRNA conformation seems to be maintained during the whole process.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2005. , p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 44
Keyword [en]
Molecular biology, trans-translation, tmRNA, SmpB, kinetics, footprinting
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-5756ISBN: 91-554-6226-X (print)OAI: oai:DiVA.org:uu-5756DiVA, id: diva2:166277
Public defence
2005-05-19, B41, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2005-04-22 Created: 2005-04-22Bibliographically approved
List of papers
1. Pre-binding of small protein B to a stalled ribosome triggers trans-translation
Open this publication in new window or tab >>Pre-binding of small protein B to a stalled ribosome triggers trans-translation
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2004 In: J Biol Chem, ISSN 25, Vol. 279, p. 25978-85Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92944 (URN)
Available from: 2005-04-22 Created: 2005-04-22Bibliographically approved
2. Ribosome rescue by tmRNA requires truncated mRNAs
Open this publication in new window or tab >>Ribosome rescue by tmRNA requires truncated mRNAs
2004 In: J Mol Biol, ISSN 1, Vol. 338, p. 33-41Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92945 (URN)
Available from: 2005-04-22 Created: 2005-04-22Bibliographically approved
3. tmRNA induced release of messenger RNA from stalled ribosomes
Open this publication in new window or tab >>tmRNA induced release of messenger RNA from stalled ribosomes
In: JMBArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-92946 (URN)
Available from: 2005-04-22 Created: 2005-04-22Bibliographically approved
4. Structure probing of tmRNA in distinct stages of trans-translation
Open this publication in new window or tab >>Structure probing of tmRNA in distinct stages of trans-translation
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2007 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 5, p. 713-722Article in journal (Refereed) Published
Abstract [en]

Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was                  d      dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

Keyword
tmRNA, SmpB, structural probing, lead(II), trans-translation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-92947 (URN)10.1261/rna.451507 (DOI)000245882400010 ()17400816 (PubMedID)
Available from: 2005-04-22 Created: 2005-04-22 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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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-92948 (URN)
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2010-01-13Bibliographically approved

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