CREL, THE CARBON CATABOLITE REPRESSOR PROTEIN FROM TRICHODERMA-REESEI

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated P CR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene , which was used to clone the entire gene, It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55. 6% and 54.7% overall similarity to the corresponding genes of Aspergil lus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions r ich in proline and basic amino acids, which may be involved in the int eraction with other proteins, Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was in stead identified as a domain occurring in several eucaryotic transcrip tion factors, The presence of the cre1 translation product was demonst rated with polyclonal antibodies against Cre1, which identified a prot ein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 pro tein fragment from the two zinc fingers to the region similar to the a a-sequence of eucaryotic transcription factors, was expressed in Esche richia coli as a fusion protein with glutathione S-transferase, EMSA a nd in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consen sus sequence for CreA binding (5'-SYGGRG-3'), Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this bi nding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated, We conclude that T. reesei Cre1 is the fu nctional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.