Cloning of cutinase transcription factor 1, a transactivating protein containing Cys6Zn2 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 1alpha (CTF1alpha) with a calculated molecular weight of 101,109. This protein contains a Cys6Zn2 binuclear cluster motif sharing homology to the Cys6Zn2 binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A. flavus. CTF1alpha, 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 CTF1alpha to palindrome 2. When CTF1alpha 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 1 abolished this transactivation. Thus, CTF1alpha positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter.
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Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210, USA.
Kolattukudy
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PE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D016335:Zinc Fingers
C106095:CTF1 alpha protein, Haematonectria haematococca; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; D002265:Carboxylic Ester Hydrolases; C019952:cutinase