DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.
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.
Liang
S D
SD
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Marmorstein
R
R
Harrison
S C
SC
Ptashne
M
M
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; C065682:PPR1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D015032:Zinc; D003545:Cysteine