Ligand binding to 2Î-deoxyguanosine sensing riboswitch in metabolic context.
Nucleic Acids Res
; 45(9): 5375-5386, 2017 May 19.
Article
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| MEDLINE
| ID: mdl-28115631
ABSTRACT
The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit ß of ribonucleotide reductase in Mesoplasma florum upon 2Î-deoxyguanosine binding. We characterized binding of 2Î-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) 2Î-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2Î-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 2Î-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 2Î-deoxyguanosine was not able to regulate gene expression.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Desoxiguanosina
/
Riboswitch
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2017
Tipo del documento:
Article
País de afiliación:
Alemania