CLONING OF CUTINASE TRANSCRIPTION FACTOR-1, A TRANSACTIVATING PROTEINCONTAINING CYS(6)ZN(2) BINUCLEAR CLUSTER DNA-BINDING MOTIF

Hydroxy fatty acids from plant cutin were shown previously to induce t he 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 palindro mes in this sequence, palindrome 2 was found to be essential for the i nducibility of cutinase by hydroxy fatty acids, Screening of a phage e xpression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase tran scription factor 1 alpha (CTF1 alpha) with a calculated molecular weig ht of 101,109, This protein contains a Cys(6)Zn(2) binuclear cluster m otif sharing homology to the Cys(6)Zn(2) binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. ca rlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nid ulans, and A, flavus., CTF1 alpha, expressed in Escherichia coli, show ed specific binding to the palindrome 2 DNA fragment but not to palind rome 1 or mutant palindrome 2 DNA fragments, suggesting specific bindi ng of CTF1 alpha to palindrome 2. When CTF1 alpha was expressed as a f usion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramph enicol acetyl transferase (cat) gene, Mutation of palindrome 2 but not palindrome I abolished this transactivation. Thus, CTF1 alpha positiv ely acts in vivo by binding selectively to palindrome 2 of the cutinas e gene promoter.