Metal-responsive elements in Pleurotus ostreatus laccase gene promoters.
Fungal laccase gene transcription is strongly induced by copper ions; notably, some laccase promoters contain multiple putative metal-responsive elements (MREs). Previously, it has been demonstrated that the Pleurotus ostreatus laccase genes poxc and poxa1b are transcriptionally induced by copper, and several putative MREs were found in the promoter regions of these genes, which extend for about 400 nt upstream of the start codon (ATG). Identification of MRE sequences, which are protected by protein binding in the poxc and poxa1b promoter regions, has been achieved by footprinting analyses. Electromobility shift assays led to the evaluation of the ability of the identified MREs to bind protein(s), and the role of specific nucleotides of these elements in complex formation has also been analysed. The formation of complexes between analysed MREs and fungal proteins requires the absence of metal ions. Proteins extracted from fungus grown in copper-depleted medium are able to form complexes with MREs, whilst proteins extracted from fungus grown in copper-containing medium are able to form complexes only in the presence of a metal chelator. Moreover, copper-depleted proteins are unable to form complexes when copper or zinc ions are added. UV-cross-linking analyses led to the determination of the molecular masses of the MRE-binding proteins. In the poxa1b promoter, a GC-rich region, homologous to the core binding site for transcription factor Sp1, decreases the binding affinity of the adjacent MRE, affecting its interactions with fungal protein factors.
Faraco
Vincenza
V
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cinthia, I-80126 Napoli, Italy.
Giardina
Paola
P
Centro Regionale di Competenza Applicazioni Tecnologico-Industriali Di Biomolecole E Biosistemi, Regione Campania, Italy.
Sannia
Giovanni
G
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cinthia, I-80126 Napoli, Italy.
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005800:Genes, Fungal; D042845:Laccase; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D016329:Sp1 Transcription Factor; D010088:Oxidoreductases; D042845:Laccase