Abstract
In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.
Highlights
Baker’s yeast Saccharomyces cerevisiae contains a family of eight basic region-leucine zipper transcription factors involved in the response to stress, designated as the Yap family [1]
The basic region-leucine zipper (bZIP) transcription factors contain a region rich in basic amino acids that is relevant to the specificity and affinity of these regulators to DNA [4,18,21]
The yeast bZIP transcription factor Yap8 plays a pivotal role in the response to arsenic stress, being the major regulator of the arsenate reductase (ACR2) and arsenite efflux pump gene (ACR3) [8,9,11,25]
Summary
Baker’s yeast Saccharomyces cerevisiae contains a family of eight basic region-leucine zipper (bZIP) transcription factors involved in the response to stress, designated as the Yap family [1]. The best studied member, is activated by thiol reactive chemicals and peroxides, which leads to its nuclear sequestration [2]. This transcription factor was identified due to its ability to bind a DNA sequence containing the simian virus 40 (SV40) sequence TGACTAA [3]. Later Yap was shown to bind more efficiently the sequence TTACTAA, that is here designated Yap response element (YRE) [4]. Yap is able to bind the sequences TGACTAA, TTAGTCA and T(T/G)ACAAA [6] [7]
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