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Lead(II) as a Tool for Probing RNA Structure in vivo
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Blyjoner som ett verktyg för att undersöka RNA strukturen in vivo (Swedish)
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. , p. 74
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 49
Keywords [en]
Molecular biology, lead(II) cleavage, RNA structure, in vivo probing
Keywords [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-5780ISBN: 91-554-6239-1 (print)OAI: oai:DiVA.org:uu-5780DiVA, id: diva2:166358
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
List of papers
1. Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation
Open this publication in new window or tab >>Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation
2001 (English)In: Antisense Nucleic Acid Drug Dev, Vol. 11, no 1, p. 29-40Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93007 (URN)
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
2. Lead(II) as a probe for investigating RNA structure in vivo
Open this publication in new window or tab >>Lead(II) as a probe for investigating RNA structure in vivo
2002 (English)In: RNA, Vol. 8, no 4, p. 534-541Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93008 (URN)
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
3. A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens
Open this publication in new window or tab >>A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens
2004 (English)In: J Biol Chem, Vol. 279, no 24, p. 25066-25074Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93009 (URN)
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
4. Lead(II) cleavage analysis of RNase P RNA in vivo
Open this publication in new window or tab >>Lead(II) cleavage analysis of RNase P RNA in vivo
(English)Article in journal (Refereed) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93010 (URN)
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
5. 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
Show others...
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.

Keywords
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

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