The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

Abstract

The repressible acid phosphatase gene PHOS of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHOS gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHOS promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PH04 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASpl) and the other located at -239 to -262 (UASp2). Exonuclease m footprint experiments revealed stops at -349 and -368 (UASpl) as well as at -245 and -260 (UASp2). Gel retardation assays with the PH02 protein revealed a binding region that lay between the two PH04-binding sites. DNase I footprint analysis suggested a PH02-binding site covering the region between -277 and -296.The promoter region of the strongly regulated acid phosphatase gene PH05 (4) has been recently analyzed by deletion mapping (6,19,24). In addition to the TATA box, two regions were found (24) to be essential for activation of the PHOS promoter. Sequence comparisons defined four elements from which a 19-base-pair dyad consensus sequence was deduced. Three of the elements, called UASp, were located within deletions showing reduced transcription of PHOS (24), at -367, -245, and -185 relative to the translational start.Not much is known about the physical interaction of regulatory proteins with the PHOS promoter. Since mutations defective in PH02 or PH04 are epistatic to all other regulatory mutations and show a negative phenotype, it is likely that the PHO2 and PHO4 proteins are positive transcription factors that interact with the PHOS promoter DNA. The finding that a pho2 mutation can be complemented by overexpression of the PHO4 protein (20) is a further indication that the PHO2 and PHO4 proteins exert their functions at the same late hierarchical level of PH05 activation.Extensive studies of the chromatin fine structure of the PH05 gene (1, 2, 5, 7) have been done. Interestingly, the element at -367, which is essential for gene induction (24), resides in the center of a hypersensitive region under conditions of PH05 repression (1, 2).In press). Interestingly, the pho2 mutation showed no influence on the basal level of TRP4 expression but rather modulated the general control response of TRP4.MATERIALS AND METHODS Escherichia coli vector for PH04 and PH02 expression. The PH04 gene (13, 15) was reisolated from a S288C background by screening an ordered centromere library (25). An NcoI site at the ATG of PH04 was introduced by site-specific mutagenesis (14). The PH04 gene (NcoI-BamHI fragment) was placed under control of the inducible PL promoter by using the vector pPLmu, a derivative of PLc24 (23) containing the phage Mu Ner gene ribosome-binding site with an NcoI site at its ATG start codon (obtained from Biogen).The PH02 gene (25) was fused to the same vector but carrying a differ...

DNA binding site of the yeast heteromeric Ino2p/Ino4p basic helix‐loop‐helix transcription factor: structural requirements as defined by saturation mutagenesis

“…Otherwise, position N~I resembles N~ in being obviously not very critical, either. A similar deviation from the CANNTG rule has been observed for one of the Pho4p binding sites in the upstream region of the acid phosphatase gene PH05 [13]. While the bHLH protein Pho4p binds efficiently to a CACGTG core element (UASp2) of the PH05 promoter, the sequence CACGTT (UASp0 is recognized with only moderate affinity.…”

Negatively cis-acting elements in the distal part of the promoter of Epstein--Barr virus trans-activator gene BZLF1

“…These are much more sensitive than DNase I assays (Vogel et al, 1989) because each bound protein molecule theoretically yields a positive signal. The results of the footprint assays are depicted in Fig.…”

Clustering Genes Using Heterogeneous Data Sources

“…YBR093C, which, according to TRANSFAC (Matys, Fricke et al 2003), is known to be the binding site for the transcription factor PHO4 and has been reported previously in the literature (Vogel, Horz et al 1989). Genes that shared this motif are YBR093C, YAR018C, YDR055W, YHR215W, YML034W, and YOR313C.…”