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Ligand binding to 2'-deoxyguanosine sensing riboswitch in metabolic context.

Kim, Yong-Boum and Wacker, Anna and Laer, Karl von and Rogov, Vladimir V. and Suess, Beatrix and Schwalbe, Harald (2017):
Ligand binding to 2'-deoxyguanosine sensing riboswitch in metabolic context.
In: Nucleic acids research, 45 (9), pp. 5375-5386. ISSN 1362-4962,
[Article]

Abstract

The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit β of ribonucleotide reductase in Mesoplasma florum upon 2'-deoxyguanosine binding. We characterized binding of 2'-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and 'in-cell-like' conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. 'In-cell-like' environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under 'in-cell-like'-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2'-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2'-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2'-deoxyguanosine, guanosine plays a role in riboswitch function in vivo Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2'-deoxyguanosine was not able to regulate gene expression.

Item Type: Article
Erschienen: 2017
Creators: Kim, Yong-Boum and Wacker, Anna and Laer, Karl von and Rogov, Vladimir V. and Suess, Beatrix and Schwalbe, Harald
Title: Ligand binding to 2'-deoxyguanosine sensing riboswitch in metabolic context.
Language: English
Abstract:

The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit β of ribonucleotide reductase in Mesoplasma florum upon 2'-deoxyguanosine binding. We characterized binding of 2'-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and 'in-cell-like' conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. 'In-cell-like' environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under 'in-cell-like'-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2'-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2'-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2'-deoxyguanosine, guanosine plays a role in riboswitch function in vivo Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2'-deoxyguanosine was not able to regulate gene expression.

Journal or Publication Title: Nucleic acids research
Journal volume: 45
Number: 9
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 13 Feb 2017 11:41
Identification Number: pmid:28115631
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