Two adjacent protein binding motifs in the cbh2 (cellobiohydrolase II-encoding) promoter of the fungus Hypocrea jecorina (Trichoderma reesei) cooperate in the induction by cellulose

The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoder ma reesei) consists of several cellobiohydrolases, endoglucanases, and beta -glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disaccharide sophorose, Using cell-free extracts from sophorose-induced and noninduced mycelia and various fragmen ts of the cbh2 promoter of H. jecorina in electrophoretic mobility shift as say (EMSA) analysis and performing in vitro and in vivo footprinting analys is, we detected the nucleotide sequence 5'-ATTGGGTAATA-3' (consequently nam ed cbh2-activating element (CAE)) to bind a protein complex with different migration in EMSA of induced and noninduced cell-free extracts. EMSA analys is, employing oligonucleotide fragments containing specifically mutated ver sions of CAE, revealed that protein binding requires the presence of an int act copy of either one of two adjacent motifs: a CCAAT (=ATTGG) box on the template strand and a GTAATA box can the coding strand, whereas a simultane ous mutation ill both completely abolished binding. H. jecorina transforman ts, containing correspondingly mutated versions of the cbh2 promoter fused to the Escherichia coil hph gene as a reporter, expressed hph in a manner p aralleling the efficacy of CAE-protein complex formation in EMSA, suggestin g that the presence of either of both motifs is required for induction of c bh2 gene transcription. Antibody supershift experiments with anti-HapC anti serum as well as EMSA competition experiments with CCAAT binding promoter f ragments of the Aspergillus nidulans amdS promoter suggest that the H. jeco rina CCAAT box binding complex contains a homologue of HapC, The nature of the adjacent, GTAATA-binding protein(s) and its cooperation with the HapC h omologue in cbh2 gene induction is discussed.