Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

Abstract

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 high-affinity binding of SW15 to this site. We show that the two proteins bind DNA cooperatively, and we take advantage of this cooperativeness to purify the stimulatory factor. In another report (5a), we demonstrate that this stimulatory factor is encoded by the GRFJO gene, and therefore, we will refer to this factor that stimulates SWI5 binding as GRF10. The GRF10 gene is also known as PH02 and BAS2. PH02 was identified as a transcriptional activator of PH05 (33), and BAS2 is required for the basal transcriptional activation of the HIS4 gene (3). The name GRF1O (for general regulatory factor) was chosen as a new gene designation since GRFIO plays a role in the transcriptional regulation of diverse genes (49). GRF1O is also required for the full expression of the HO gene (5a).MATERIALS AND METHODS Yeast strains. Yeast strain DY411 (A Ta swiS::hisG ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52) is a derivative of DY150 (9) which contains a hisG (2) disruption of SWI5. DY472 is strain DY411 which contains plasmid YCp5O:pGAL:SfW5, which overexpresses SWI5 (43)

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.…”

“…The results of electrophoretic mobility shift assays suggested that BAS2 binds to the ADE2 and ADE5,7 promoters; however, no specific binding sites were identified (3). 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).…”

“…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).…”

“…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). BAS2 also interacts with PHO4 to induce PHO5 transcription under phosphate starvation conditions (16).…”

In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

“…Furthermore, cleavage by thrombin releases the DNA-binding protein (17). The GST fusion protein system was successfully used to determine the DNA-binding sites for a number of proteins such as SWI5 in yeast (18), N-Myc in mouse (19), and T/E1A in human (20). In A. nidulans, binding sites for the CreA repressor (16) and NirA (21) and UaY (6) activators have been localized using GST fusion proteins.…”