DNA Sequence Preferences of GAL4 and PPR1: How a Subset of Zn₂Cys₆ Binuclear Cluster Proteins Recognizes DNA

Abstract

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5 to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.GAL4 and PPR1 (pyrimidine pathway regulator 1), two Saccharomyces cerevisiae transcription regulatory proteins, are members of a family of at least 12 fungal transcription factors containing a region termed the Zn 2 Cys 6 binuclear cluster (reviewed in reference 11). This region has six absolutely conserved cysteines and a number of other highly conserved residues. It has been shown for various members of the family that the Zn 2 Cys 6 region is essential for DNA binding (reviewed in references 15 and 24). The DNA sites of several members of the family, including those recognized by GAL4 and PPR1, contain palindromic CGG triplets within their DNA sites (Fig. 1a). Thus, GAL4 and PPR1 are members of a subset of Zn 2 Cys 6 -containing proteins whose members recognize twofold symmetric CGG half sites separated by distinct numbers of base pairs (24,29).GAL4 is an activator of transcription of various galactoseinducible genes. These genes possess GAL4 binding sites positioned in the upstream regions of their promoters (3, 4, 9). Examination of 16 known natural sites reveals a 17-bp pseudopalindromic consensus sequence (9). This consensus site is a high-affinity binding site for GAL4 derivatives in vitro (this study) and confers GAL4-dependent transcriptional activation in vivo when placed upstream of a test reporter gene, both in yeast cells (18, 21a) and in mammalian tissue culture cells (36). The site has highly co...

Transcriptional Repression by v-Ski and c-Ski Mediated by a Specific DNA Binding Site

“…A HindIII to BglII fragment of the pG5tkCAT plasmid (26), in which the HindIII site had been filled in using dATP and dGTP only, containing the TK promoter and the five Gal4 binding sites, was cloned into NheI to BglII-digested pGL3 Basic in which the NheI site had been filled in using dCTP and dTTP only. To eliminate a putative Gal4 binding site in the luciferase cassette (27), a HindIII to BsrG1 segment of pGL3 Basic was replaced with a similar fragment from pGL2 Basic.…”

Alternative Mechanisms of Transcriptional Activation by Rap1p

“…Fig. 2 shows that, when transformed into BY4741 cells, this construct behaved as a typical UASgal reporter, silent in glucose (about 3 Miller units) and strongly activated in galactose (over 250 units (48)). Conversely, plasmid pGRC5 gave a weak activation in glucose, as a single Rap1p-binding site (around 25 units (5)).…”

“…2). Note that this residue lies outside the Gal4p-binding sequence in a position that does not affect Gal4p binding (48). Fig.…”

A nucleosome positioned by α2/Mcm1 prevents Hap1 activator binding in vivo

“…Both Gal4 and Hap1 have the Zn 2 Cys 6 DNA binding motif and bind as dimers, but Gal4 and Hap1 recognize two CGG triplets symmetrically and asymmetrically, respectively. In addition, the space between the two triplets, which is important for their affinity [36], is different between Gal4 and Hap1. Thus, the configuration of the CGG triplets in the nucleosome may affect their recognition ability.…”