Binding and activation by the zinc cluster transcription factors of Saccharomyces cerevisiae: redefining the UASGABA and its interaction with Uga3p

摘要

Uga3p, a member of zinc binuclear cluster transcription factor family, is required for γ-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Members of this family bind to CGG triplets with the spacer region between the triplets being an important specificity determinant. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCGN4CGG-everted repeat proposed to be the binding site of Uga3p, UAS GABA. The function of conserved nucleotides flanking the everted repeat has not been rigorously investigated. The interaction of Uga3p with UAS GABAwas characterized in terms of binding in vitro and transcriptional activation of lacZ reporter genes in vivo. Electromobility shift assays using mutantUAS GABA sequences and heterologously produced full-length Uga3p demonstrated that UAS GABAconsists of two independent Uga3p binding sites. Simultaneous occupation of both Uga3p binding sites of UAS GABAwith high affinity is essential for GABA-dependent transcriptional activation in vivo. We present evidence that the two Uga3p molecules bound to UAS GABAprobably interact with each other and show that Uga3p(1–124), previously used for binding studies, is not functionally equivalent to the full-length protein with respect to binding in vitro. We propose that the Uga3p binding site is an asymmetric site of 5′-SGCGGNWTTT-3′ (S = G or C, W = A, or T and n = no nucleotide or G). However,UAS GABA, is a palindrome containing two asymmetric Uga3p binding sites.