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Eukaryotic RNase P RNA mediates cleavage in the absence of protein
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
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.
2007 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 7, 2062-2067 p.Article in journal (Refereed) Published
Abstract [en]

The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (k obs) was five to six orders of magnitude lower, whereas the affinity for the substrate (appK d) was reduced ≈20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.

Place, publisher, year, edition, pages
2007. Vol. 104, no 7, 2062-2067 p.
Keyword [en]
catalytic RNA, ribozyme, RNA processing
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-95832DOI: 10.1073/pnas.0607326104ISI: 000244438500008PubMedID: 17284611OAI: oai:DiVA.org:uu-95832DiVA: diva2:170185
Available from: 2007-04-27 Created: 2007-04-27 Last updated: 2011-02-11Bibliographically approved
In thesis
1. Versatile and Antique World of RNA: The Simplicity of RNA Mediated Catalysis
Open this publication in new window or tab >>Versatile and Antique World of RNA: The Simplicity of RNA Mediated Catalysis
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNA is the only biological molecule that can function both as a repository of information and as a catalyst. This, together with the ability to self-replicate, led to recognition of RNA as ‘prelude to life’.

My work highlights some of the important features of RNA as a catalyst, exemplified by RNase P. It addresses questions of evolutionary preservations of residues and structure, involvement of metal ions and finally structure evolution towards minimal catalytically competent RNA motifs.

RNase P is the only enzyme involved in 5’ end processing of all pre-tRNAs. Until recently, it was believed that the RNA moiety of RNase P is responsible for mediating catalysis only in Bacteria. However, my recent study conclusively demonstrated that eukaryotic RNase P RNA is catalytically competent in vitro in absence of proteins. These findings evidenced evolutionary preservation of RNA-mediated catalysis in RNase P.

RNase P RNA is a metalloeznyme. In my studies I analyzed the contributions of individual chemical groups at the cleavage site to catalysis. My findings suggested that the 2’OH of N-1 and the exocyclic amine of G+1 are involved in positioning of functionally important metal ions. Additionally, data appointed the function of Pb2+ as both structural metal ion and important in generating the nucleophile. My studies further indicate a conformational change upon RNase P RNA -substrate complex formation in keeping with an induced fit mechanism.

Studying the effects of reducing the ribozyme size upon dissection of bacterial RNase P RNAs, we defined the smallest catalytically competent domain i.e. P15-loop. Derivatives of this autonomous metal ion binding domain, (the smallest being 31nt-s), are able to cleave both whole-length pre-tRNAs as well as hairpin substrates, though with severely reduced rates relative to their parent ribozymes. The study has inferred that partite ES interactions at the cleavage site prove sufficient for catalysis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 298
Keyword
Microbiology, RNA Catalysis, RNase P, Metal ions, tRNA Processing, Ribozyme, Mikrobiologi
Identifiers
urn:nbn:se:uu:diva-7847 (URN)978-91-554-6879-8 (ISBN)
Public defence
2007-05-22, C10:301, Biomedical Centre, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2007-04-27 Created: 2007-04-27Bibliographically approved

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