Abstract
Members of the family of fungal zinc cluster DNA-binding proteins possess 6 highly conserved cysteines that bind to two zinc atoms forming a structure (Zn2Cys6) that is required for recognition of specific DNA sequences. Many zinc cluster proteins have been shown to bind as homodimers to a pair of CGG triplets oriented either as direct (CGG NX CGG), inverted (CGG NX CCG), or everted repeats (CCG NX CGG), where N indicates nucleotides. Variation in the spacing between the CGG triplets also contributes to the diversity of sites recognized. For example, Leu3p binds to the everted sequence CCG N4 CGG with a strict requirement for a 4-base pair spacing. Here, we show that another member of the family, Uga3p, recognizes the same DNA motif as Leu3p. However, these transcription factors have distinct DNA targets. We demonstrate that additional specificity of binding is provided by nucleotides located between the two everted CGG triplets. Altering the 4 nucleotides between to the two everted CGG triplets switches the specificity from a Uga3p site to a Leu3p site in both in vitro and in vivo assays. Thus, our results identify a new mechanism that expands the repertoire of DNA targets of the family of zinc cluster proteins. These experiments provide a model for discrimination between targets of zinc cluster proteins.
Highlights
In fungi, many transcriptional regulators are characterized by the presence of a zinc finger containing a cluster of 6 cysteines that are bound by two zinc atoms and are referred to as zinc cluster proteins or Zn2Cys6 binuclear cluster proteins
Gal4p, which is required for activation of genes related to galactose metabolism, recognizes an inverted repeat consisting of CGG triplets spaced by 11 base pairs1 (CGG N11 CCG), where N indicates nucleotides, with each zinc finger recogniz
Even though direct and inverted repeats consisting of CGG triplets are found in UASLEU3, we have shown, by mutational analysis, that Leu3p binds to an everted repeat containing two CGG triplets spaced by 4 bp (CCG N4 CGG)
Summary
Many transcriptional regulators are characterized by the presence of a zinc finger containing a cluster of 6 cysteines (consensus sequence: CX2CX6CX5–9CX2CX6–8C) that are bound by two zinc atoms and are referred to as zinc cluster proteins or Zn2Cys6 binuclear cluster proteins. Our results show that Uga3p recognizes an everted CGG repeat spaced by 4 bp, a sequence similar to the target site of Leu3p. These results show that even if the UASs of UGA1 and LEU2 are related, they are activated by distinct zinc cluster proteins, Uga3p and Leu3p, respectively.
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