Nucleic Acids Research Advance Access originally published online on August 9, 2007
Nucleic Acids Research 2007 35(16):5370-5378; doi:10.1093/nar/gkm580
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Nucleic Acids Research, 2007, Vol. 35, No. 16 5370-5378
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach
Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria
*To whom correspondence should be addressed. Tel: +43 512 507 5210; Fax: +43 512 507 2892; Email: ronald.micura{at}uibk.ac.at
Received June 20, 2007. Revised July 13, 2007. Accepted July 13, 2007.
Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures.
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