Molecular characterization of a cellulase-negative mutant of Hypocrea jecorina.
The "cbh2 activating element," CAE, consisting of two separate boxes (ATTGG = CCAAT and GTAATA, respectively) is essential for cellobiohydrolase II gene expression in the filamentous fungus Hypcrea jecorina. Here we report that cell-free extracts from a cellulase-negative mutant form CAE-protein complexes with higher mobility and lower binding-strength compared to the wild type. EMSA analysis demonstrated an increased mobility of the GTAATA-binding protein complex and, supported by in vivo footprinting, a lowered binding 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 H. jecorina, and the protein-DNA complex of the mutant shows the characteristic increase in mobility. We conclude that this mutant is defective in the functional formation of the CAE-protein complexes but not in their binding to the target sequences itself.
Zeilinger
S
S
Section Microbial Biochemistry, Institute of Biochemical Technology and Microbiology, Technical University of Vienna, Getreidemarkt 9, Wien, A-1060, Austria. szeiling@mail.zserv.tuwien.ac.at
Haller
M
M
Mach
R
R
Kubicek
C P
CP
D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D006999:Hypocreales / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism
D023081:CCAAT-Binding Factor; D005656:Fungal Proteins; D014157:Transcription Factors; C111072:aab-1 protein, Neurospora; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase