Molecular characterization of a cellulase-negative mutant of Hypocrea jecorina

The "cbh2 activating element," CAE,, consisting of two separate boxes (ATTG G = CCAAT and GTAATA, respectively) is essential for cellobiohydrolase ii g ene expression in the filamentous fungus Hypcrea jecorina. Here we report t hat cell-free extracts from a cellulase-negative mutant form CAE-protein co mplexes with higher mobility and lower binding-strength compared to the wil d type. EMSA analysis demonstrated an increased mobility of the GTAATA-bind ing protein complex and, supported by in vivo footprinting, a lowered bindi ng strength of the HAP2/3/5 proteins. However, the hap2/hap3/hap5 genes of the mutant are unaltered and transcribed normally. A nucleotide fragment of the cbh1 promoter containing a (GG)CTAATA motif without an adjacent CCAAT box is also bound by cell-free extracts of Ii. jecorina, and the protein-DN A complex of the mutant shows the characteristic increase in mobility. We c onclude that this mutant is defective in the functional formation of the CA E-protein complexes but not in their binding to the target sequences itself . (C) 2000 Academic Press.