Identification of promoter elements responsible for the regulation of from , a major facilitator transporter involved in azole resistance Free

Abstract

Upregulation of the (ultirug esistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen . To better understand the molecular mechanisms underlying the phenomenon, the -acting regulatory elements present in the promoter were characterized using a -galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or HO, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two -acting regulatory elements within the promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (). One, a enomyl esponse lement (BRE), is situated at position −296 to −260 with respect to the ATG start codon. It is required for benomyl-dependent upregulation and is also necessary for constitutive high expression of . A second element, termed O esponse lement (HRE), is situated at position −561 to −520. The HRE is required for HO-dependent upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p ( AP-1 protein) lie within the HRE. Moreover, inactivation of abolished the transient response to HO. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a -acting and positive regulatory role in the HO-dependent transcription of . A minimal BRE (−290 to −273) that is sufficient to detect sequence-specific binding of protein complexes in crude extracts prepared from was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the -acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the -acting elements of the promoter should now serve to elucidate the nature of the components that modulate its activity.

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2006-12-01
2024-03-29
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