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Lead(II) as a probe for investigating RNA structure in vivo
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2002 (English)In: RNA, Vol. 8, no 4, p. 534-541Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2002. Vol. 8, no 4, p. 534-541
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93008OAI: oai:DiVA.org:uu-93008DiVA, id: diva2:166354
Available from: 2005-04-22 Created: 2005-04-22 Last updated: 2014-05-28Bibliographically approved
In thesis
1. 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. p. 74
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 49
Keywords
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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Wagner, Gerhart

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