The Swi5 zinc-finger and Grf10 homeodomain proteins bind DNA cooperatively at the yeast HO promoter.

Abstract

SWIS encodes a zinc-finger protein required for expression of the yeast HO gene. Using SwiS protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the SwiS stimulatory factor as the product of the GRF1O gene, which encodes a yeast homeodomain protein. GRFIO, also known as PH02 and BAS2, is a transcriptional activator of the PH05 acid phosphatase gene and the HIS4 histidine biosynthesis gene. GrflO protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disasciation rates indicates that the GrflO-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or GrflO. Finally, we show that HO expression is reduced in yeast strains containing grflO null mutations and that full expression of a heterologous promoter containing a SWIS-dependent HO upstream activation sequence element requires GRFIO.

The Swi5 activator recruits the Mediator complex to theHOpromoter without RNA polymerase II

“…Swi5 and Pho2 bind to HO cooperatively (Brazas and Stillman 1993), and we identified mutations between amino acids 482 and 505 of Swi5 that affect activation of a reporter gene (Bhoite and Stillman 1998). Most of these Swi5 mutations affect its ability to bind DNA cooperatively with Pho2.…”

The phosphatase system in Saccharomyces cerevisiae.

“…Alignment of UAS sequences to which relevant regulatory proteins bind together with Pho2p. The consensus sequences in the promoter for Bas1p (Daignan-Fornier and Fink, 1992), Pho4p (Barbarić et al, 1996;Ogawa et al, 1994Ogawa et al, , 1995b, and Swi5p (Brazas and Stillman, 1993b) were as described. The core UAS sequences for binding of the indicated proteins are written in boldface letters and the regions protected in the footprinting experiments are indicated by brackets.…”

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

“…PCR reactions were performed as described above using the labeled oligonucleotides as primers and plasmids containing the appropriate ADE5,7 sequences as templates to amplify the region between Ϫ211 and Ϫ145 (relative to the ADE5,7 start codon). The templates were pR224, carrying the wild-type ADE5,7 fragment, or selected plasmids carrying mutated ADE5,7 promoter fragments, constructs numbered 5,7,10,11,13,14,19,20,27,29,34,35,39, and 43 as listed in Table IV. After PCR, a portion of each sample was separated by electrophoresis, and concentrations of the PCR products were estimated by ethidium bromide staining in comparison with known concentrations of duplex oligonucleotides of approximately the same length.…”

“…In addition to participating with BAS1 in transcriptional activation of HIS4 and the ADE genes, BAS2 (also known as PHO2 and GRF10) stimulates transcription of PHO5 (12) and HO (13). High level transcription of HO requires BAS2 in conjunction with SWI5 (13,14). Binding of BAS2 and SW15 to the HO promoter is cooperative in vitro (13,15).…”