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Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach
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 ch...
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018614/ https://www.ncbi.nlm.nih.gov/pubmed/17693433 http://dx.doi.org/10.1093/nar/gkm580 |
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author | Lang, Kathrin Rieder, Renate Micura, Ronald |
author_facet | Lang, Kathrin Rieder, Renate Micura, Ronald |
author_sort | Lang, Kathrin |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2018614 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-20186142007-10-23 Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach Lang, Kathrin Rieder, Renate Micura, Ronald Nucleic Acids Res RNA 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. Oxford University Press 2007-08 2007-08-09 /pmc/articles/PMC2018614/ /pubmed/17693433 http://dx.doi.org/10.1093/nar/gkm580 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ 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. |
spellingShingle | RNA Lang, Kathrin Rieder, Renate Micura, Ronald Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title_full | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title_fullStr | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title_full_unstemmed | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title_short | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach |
title_sort | ligand-induced folding of the thim tpp riboswitch investigated by a structure-based fluorescence spectroscopic approach |
topic | RNA |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018614/ https://www.ncbi.nlm.nih.gov/pubmed/17693433 http://dx.doi.org/10.1093/nar/gkm580 |
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