A Novel DNA Binding Motif for Yeast Zinc Cluster Proteins: the Leu3p and Pdr3p Transcriptional Activators Recognize Everted Repeats

Abstract

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.

Multiple Yap1p-binding Sites Mediate Induction of the Yeast Major Facilitator FLR1 Gene in Response to Drugs, Oxidants, and Alkylating Agents

“…Cells were lysed by glass-bead disruption as described previously (39) and centrifuged at 12,000 ϫ g for 1 h at 4°C. The supernatants were harvested, and the proteins were quantified by the method of Bradford (37).…”

Yeast Transcriptional Regulator Leu3p

“…Leu3p exists as a dimer, both in the presence and absence of DNA (6,7). It recognizes the sequence 5Ј-CCGN 4 CGG-3Ј (8). Binding of full-length Leu3p causes the target DNA to bend, with an apparent flexure angle of 46 o .…”

Mode of AmyR Binding to the CGGN8AGG Sequence in theAspergillus oryzaetaaG2Promoter

“…A subset of the DNA-binding domains with this motif has been shown to bind inverted, everted, or direct repeats of a CGG triplet. [9][10][11][12][13][14] A. oryzae AmyR binds two types of sequences; one is characterized by two CGG triplets separated by eight nucleotides, and the other is 5 0 -CGGAAATTTAA-3 0 , which corresponds exactly to SRE in the taaG2 promoter. 5,8) The amyR gene was also cloned from A. nidulans.…”