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Metal ion binding to group II intron ribozymes : a study from bulk to the single molecule level


Steiner, M. Metal ion binding to group II intron ribozymes : a study from bulk to the single molecule level. 2008, University of Zurich, Faculty of Science.

Abstract

This work combines three approaches to study metal ion interactions with ribonucleic acid (RNA). The model systems used are large catalytic RNAs known as group II intron ribozymes. First, metal ion binding sites were located on the ribozyme sequence Av.D135 using the Tb3+-cleavage technique and the results were compared to an analogous study on the ribozyme Sc.D135. Second, the influence of binding of several metal ions on the local bulge structure of domain 5 (D5, a ribozyme subelement) was studied. The D5 bulge becomes unstacked upon binding of all divalent metal ions tested as indicated by the increasing fluorescence of the 2-aminopurine reporter group located in the bulge. The last section - the highlight in this work - describes a colorful fluorescence study of the structure and dynamics of the multidomain molecules Sc.D135 at the single molecule fluorescence level. The riboyzmes show three individual conformational states along the folding pathway that are in dynamic equilibrium connected by small energy barriers. With increasing Mg2+ concentrations, the molecule's dynamics increase, enabling the ribozyme to reach the active state. The results permit the characterization of distinct effects of individual metal ions on RNA and imply a new paradigm for large RNA folding.

Abstract

This work combines three approaches to study metal ion interactions with ribonucleic acid (RNA). The model systems used are large catalytic RNAs known as group II intron ribozymes. First, metal ion binding sites were located on the ribozyme sequence Av.D135 using the Tb3+-cleavage technique and the results were compared to an analogous study on the ribozyme Sc.D135. Second, the influence of binding of several metal ions on the local bulge structure of domain 5 (D5, a ribozyme subelement) was studied. The D5 bulge becomes unstacked upon binding of all divalent metal ions tested as indicated by the increasing fluorescence of the 2-aminopurine reporter group located in the bulge. The last section - the highlight in this work - describes a colorful fluorescence study of the structure and dynamics of the multidomain molecules Sc.D135 at the single molecule fluorescence level. The riboyzmes show three individual conformational states along the folding pathway that are in dynamic equilibrium connected by small energy barriers. With increasing Mg2+ concentrations, the molecule's dynamics increase, enabling the ribozyme to reach the active state. The results permit the characterization of distinct effects of individual metal ions on RNA and imply a new paradigm for large RNA folding.

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Additional indexing

Item Type:Dissertation
Referees:Sigel R K O, Rueda D, Berke H G
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2008
Deposited On:02 Mar 2009 12:04
Last Modified:06 Dec 2017 18:54
Number of Pages:168
Official URL:http://opac.nebis.ch/exlibris/aleph/a18_1/apache_media/KDDM2KDUD1JR5HNS2GV1I6CDNTXLHH.pdf
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005656385

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