The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element.
The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.
Zeilinger
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Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, Technische Universität Wien, Austria. szeiling@mail.zserv.tuwien.ac.at
Ebner
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Marosits
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Mach
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Kubicek
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D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000235:genetics*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D017931:DNA Primers; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006999:Hypocreales / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D023081:CCAAT-Binding Factor; D017931:DNA Primers; D005656:Fungal Proteins; C437709:HAP2 protein, Hypocrea jecorina; C437710:HAP3 protein, Hypocrea jecorina; C437711:HAP5 protein, Hypocrea jecorina; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase