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Thermodynamic characterization of an engineered tetracycline-binding riboswitch.

Müller, Michael and Weigand, Julia E. and Weichenrieder, Oliver and Suess, Beatrix (2006):
Thermodynamic characterization of an engineered tetracycline-binding riboswitch.
In: Nucleic acids research, pp. 2607-17, 34, (9), ISSN 1362-4962,
[Article]

Abstract

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

Item Type: Article
Erschienen: 2006
Creators: Müller, Michael and Weigand, Julia E. and Weichenrieder, Oliver and Suess, Beatrix
Title: Thermodynamic characterization of an engineered tetracycline-binding riboswitch.
Language: English
Abstract:

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

Journal or Publication Title: Nucleic acids research
Volume: 34
Number: 9
Divisions: 10 Department of Biology > Synthetic Genetic Circuits
10 Department of Biology
Date Deposited: 22 Feb 2012 10:35
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