Submitted to: Gordon Research Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: July 24, 1998
Publication Date: N/A
Technical Abstract: Trichothecene biosynthetic pathway genes are localized within a gene cluster in F. sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding in vitro. Analysis of TRI6 binding within the TRI3 and TRI11 promoters, and the TRI4-TRI6 intergenic region which do not contain the TNAGGCCT motif, suggests that the minimum sequence for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, TRI4-A and TRI4-B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the TRI4-A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4P/GUS fusions. Substitution of an Ala residue for Cys187 within the predicted C2H2 zinc-finger motif at the C-terminus of TRI6 eliminated DNA binding activity in vitro and supports the involvement of Cys187 in DNA binding. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted C2H2 zinc fingers.