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Structure probing of tmRNA in distinct stages of trans-translation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Ehrenberg)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Ehrenberg)
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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, 713-722 p.Article 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.

Place, publisher, year, edition, pages
2007. Vol. 13, no 5, 713-722 p.
Keyword [en]
tmRNA, SmpB, structural probing, lead(II), trans-translation
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-92947DOI: 10.1261/rna.451507ISI: 000245882400010PubMedID: 17400816OAI: oai:DiVA.org:uu-92947DiVA: diva2:166275
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
In thesis
1. Finding the unknowns in trans-translation
Open this publication in new window or tab >>Finding the unknowns in trans-translation
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Hitta de okända faktorerna för trans-translation
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. 69 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 44
Keyword
Molecular biology, trans-translation, tmRNA, SmpB, kinetics, footprinting, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-5756 (URN)91-554-6226-X (ISBN)
Public defence
2005-05-19, B41, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2005-04-22 Created: 2005-04-22Bibliographically approved
2. Lead(II) as a Tool for Probing RNA Structure in vivo
Open this publication in new window or tab >>Lead(II) as a Tool for Probing RNA Structure in vivo
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Blyjoner som ett verktyg för att undersöka RNA strukturen in vivo
Abstract [en]

Chemical modification and limited enzymatic hydrolysis are powerful methods to obtain detailed information on the structure and dynamics of RNAs in solution. In the work presented here I have taken advantage of the properties of the divalent metal ion lead(II) to establish it as a new probe for investigating the structure of RNA in vivo. Besides highly specific lead(II)-induced cleavage due to the presence of tight metal ion binding sites, lead(II) is known to cleave RNA within single-stranded regions, loops and bulges. The detailed structural data obtained with three different RNAs: tmRNA, CopT, and the leader region of the ompF mRNA, show that lead(II) has great potential for in vivo studies of RNA structure. In P. fluorescens, the activity and stability of RsmY, a small regulatory RNA, was shown to be strongly dependent on repeated GGA motifs in single-stranded regions. In vivo lead(II) probing essentially confirmed predicted secondary structures and also indicated binding to a protein, RsmA. The potential in using lead(II) for mapping protein binding sites on RNAs was shown for the interaction between E. coli tmRNA and the SmpB protein. In vivo and in vitro data show protections in the tRNA-like domain of tmRNA due to binding to the SmpB protein, indicating that the SmpB protein is associated with the majority of tmRNA in the cell.

Furthermore, the overall conformation/ structure of E. coli RNase P was analyzed by probing the native structure of M1 RNA in vivo with lead(II). The observed cleavages suggests that M1 RNA is present in two main conformations in the cell, one being characteristic of free RNase P, and one of an RNase P-tRNA complex. The results also indicate that the C5 protein subunit has only minor effects on the overall structure of the RNA subunit.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 49
Keyword
Molecular biology, lead(II) cleavage, RNA structure, in vivo probing, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-5780 (URN)91-554-6239-1 (ISBN)
Public defence
2005-05-14, Room B41, BMC, Husargatan 3, Uppsala, 11:00 (English)
Opponent
Supervisors
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2010-02-22Bibliographically approved

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Pavlov, MichaelWagner, Gerhart E. H.Ehrenberg, Måns

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