Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.

Abstract

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequencespecific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: C+8GCCMl . addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

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

“…These results are in agreement with previously published data on the Kluyveromyces lactis GAL4 homolog, LAC9 (10). They are also in accord with the structure of GAL4-DNA complex (24), which reveals base-pair-specific contacts to CGG triplet sequences at the ends of the DNA site.…”

The Regulator of Nitrate Assimilation in Ascomycetes Is a Dimer Which Binds a Nonrepeated, Asymmetrical Sequence

“…The pattern of methylation interference clearly differs from that found in proteins recognizing the CGG motif. For both Lac9p and UaY, the only base where methylation interferes with binding is the second G (13,32). This is the only G whose N7 accepts a proton from the ε-NH 2 group of a conserved lysine in the structures of the Gal4p and Ppr1p protein-DNA complexes (24,25).…”

“…Thus, it is the nature of this linker that determines the specificity of binding (23,29). The differences between Gal4p and Lac9p, on the one hand, and Ppr1p and UaY, on the other, are accounted for by the fact that the linker of the latter forms a ␤-sheet which shortens the distance between the dimerization element and the DNA binding domain (13,24,25,32). Recently, it has been shown that Cyp1p (Hap1p) recognizes a direct repeat of the same base composition as those of the above proteins and that Leu3p and probably Pdr3p recognize identical everted repeats (14,36).…”

Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine.

“…Comparing known target sites of the yeast regulatory proteins of the C6 family (Table 4), we realized that they all share such a conserved CGPu sequence that we propose to call CEZ (contributing element for binding of C6 zine clusters). A recent study of DNA binding by the LAC9 protein has shown that the CGG nucleotides at both ends of the 17-mer UAS are essential (26). Moreover, it is possible that the CGPu motif is also involved in the recognition of the C2H2 type of zinc finger; indeed, it is present in the minor region in SWI5 that is protected against DNase, in the DNA region involved in the ADR1 response (but not in the 22-bp palindrome), in GC boxes recognized by Spl, in the ICR control region governing TFIIIA binding, and in the MRE sequence bound by MTF1 (Table 4).…”

“…The HAP1 protein is the only other member of the C6 zinc cluster family of proteins known to bind to apparently asymmetrical DNA sequences (the B region of CYCI UAS1 and the unrelated tandemly repeated 9-bp sequence found in the UAS in front of CYC7) (23,24,61). (34) GAL2 (34) MELI (34) LAC4 (26,82) URA3 (69) URAI (69) URA4 (69) PUTi (72) PUT2 (72 [59]) and the mammalian serum-responsive factor (SRF; 40 identities [58]). …”