Cloning of cutinase transcription factor 1, a transactivating protein containing Cys(6)Zn(2) binuclear cluster DNA-binding motif

Hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at -159 base pairs of the cutinase gene in Fusarium solani f. sp, pisi (Nectria hematococca mating type VI), Of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids, Screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase transcription factor 1 alpha (CTF1 alpha) with a calculated molecular weight of 101,109, This protein contains a Cys(6)Zn(2) binuclear cluster motif sharing homology to the Cys(6)Zn(2) binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A, flavus., CTF1 alpha, expressed in Escherichia coli, showed specific binding to the palindrome 2 DNA fragment but not to palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific binding of CTF1 alpha to palindrome 2. When CTF1 alpha was expressed as a fusion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramphenicol acetyl transferase (cat) gene, Mutation of palindrome 2 but not palindrome I abolished this transactivation. Thus, CTF1 alpha positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter.