RESUMEN
We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.
Asunto(s)
Magnesio/metabolismo , ARN/metabolismo , Tiamina Pirofosfato/metabolismo , Regiones no Traducidas 5' , Adenosina/metabolismo , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , Secuencia Conservada , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Cinética , Magnesio/farmacología , Magnesio/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , ARN/química , ARN/clasificación , ARN/genética , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido Nucleico , Temperatura , Volumetría , Transcripción GenéticaRESUMEN
Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.