The Gal4, Put3, and Ppr1 yeast zinc cluster proteins bind as homodimers to DNA sequences composed of palindromic CGG triplets. Spacing between the triplets specifies the target site for a given zinc cluster protein. In addition, Hap1p, another zinc cluster protein, also recognizes CGG triplets but only when oriented as a direct repeat. Unexpectedly, our results show that Leu3p, another member of this family, also recognizes CGG triplets but oriented in opposite directions and spaced by 4 nucleotides (an everted repeat or inverted palindrome: CCG-N4-CGG). This constitutes a novel DNA motif for zinc cluster proteins. Moreover, the presence of this motif was shown to be essential for in vivo activation by Leu3p of a minimal reporter containing one copy of a target site for this activator. We also provide evidence that another member of this family, Pdr3p, binds to an everted repeat spaced by 0 nucleotides (CCGCGG). Thus, our results show that three CGG motifs are used by members of the zinc cluster family: palindromes, direct repeats, and everted repeats.
The yeast PDR5 gene encodes an efflux pump that confers multidrug resistance. Expression of PDR5 is positively regulated by the transcription factors Pdr1p and Pdr3p that recognize the same pleiotropic drug resistance elements (PDREs) in the PDR5 promoter. Pdr1p and Pdr3p belong to the Gal4p family of zinc cluster proteins. The function of RDR1 (YOR380W), which also encodes a member of this family, is unknown. To identify target genes for Rdr1p, we have performed wholegenome analysis of gene expression with DNA microarrays. Our results show that Rdr1p is a transcriptional repressor of five genes, including PDR5. A ⌬rdr1 strain has increased resistance to cycloheximide, as expected from the overexpression of PDR5. In addition, the activity of a PDR5-lacZ reporter is increased in a ⌬rdr1 strain. All (but one) genes affected by removal of Rdr1p contain PDREs in their promoters. We tested if the effect of Rdr1p is mediated through PDREs by inserting this DNA element in front of a minimal promoter. Activity of this reporter was increased in a ⌬rdr1 strain. Moreover, mutations known to reduce binding of Pdr1/ Pdr3p abolished the induction observed in the ⌬rdr1 strain. Thus, we have identified a transcriptional repressor involved in the control of multidrug resistance.Multidrug resistance is a widespread phenomenon that allows organisms ranging from bacteria to humans to defend themselves against a variety of toxic compounds. Drug resistance is mediated through membrane-bound transporters that act as drug efflux pumps. This process has been extensively studied in the yeast Saccharomyces cerevisiae. Two major classes of multidrug transporters have been identified: the major facilitator superfamily (MFS) 1 (1) and the ABC (ATP binding cassette) family of transporters (2-4). ABC transporters act in an ATP-dependent manner, and their overexpression leads to increased drug resistance. Members of the family of ABC transporters include Pdr5p, Snq2p, and Yor1p (2).The transcriptional activators Pdr1p and Pdr3p control the expression of many ABC transporters (3, 4). These activators belong to a family of transcriptional regulators called zinc cluster or binuclear cluster proteins (5-7). Members of this family contain six highly conserved cysteines that coordinate binding to two zinc atoms to allow proper folding of the DNA binding domain. The cysteine-rich region (or zinc finger) is usually followed by a short linker sequence that bridges the zinc finger to a dimerization domain (6). This domain is involved in recognition of specific DNA sequences through interaction of the zinc finger with DNA (for references see Ref. 8). Pdr1p and Pdr3p both recognize CGG triplets oriented in opposite directions (CCGCGG) to form an everted repeat (9). This motif is found in the target genes of Pdr1p and Pdr3p (10 -17). For example, three binding sites called PDREs (pleiotropic drug response elements) for Pdr1p and Pdr3p have been mapped in the PDR5 promoter (10, 12). Thus, Pdr1p and Pdr3p recognize the same DNA sequences in target promoters f...
The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site-directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1.
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