Determining the Requirements for Cooperative DNA Binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO Promoter

Abstract

SW15 encodes a zinc finger DNA binding protein required for the transcription of the Saccharomyces cerevisiae HO gene, and PHO2 encodes a homeodomain DNA binding protein. In vitro biochemical studies using purified Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p bind cooperatively to the HO promoter. In this report we investigate the regions of the Swi5p and Pho2p proteins required for cooperative DNA binding. The analysis of each protein gives a similar result: the zinc finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are required for cooperative DNA binding. In vitro and in vivo experiments were conducted with promoters with altered spacing between the Pho2p and Swi5p binding sites. Mutations that increased the distance between the two binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity. These observations suggest that the interaction domains of Swi5p and Pho2p are flexible and can tolerate an increase in distance between the two binding sites. The mechanism of the cooperative DNA binding by Swi5p and Pho2p is discussed.

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

“…5A, lanes 1, 5, and 6). In an effort to verify these results, we carried out EMSA experiments comparing wild-type and mutant competitors using a purified polyhistidine-tagged form of BAS2 expressed in E. coli (13,15). We saw small reductions in the ability of fragments containing mutations in clusters 15 and 16 or clusters 18 and 19 relative to the wild-type ADE5,7 fragment to compete with the labeled wildtype probe for (His) 6 -BAS2 binding (data not shown).…”

“…The boldface type corresponds to the regions discussed in the text: region I, clusters 2-6; region II, clusters 10 -13; region III, clusters 15-16; region IV, clusters 18 -20. The 5Ј-most nucleotide in each cluster is as follows: cluster 1, Ϫ211; cluster 2, Ϫ208; cluster 3, Ϫ205; cluster 4, Ϫ202; cluster 5, Ϫ199; cluster 6, Ϫ196; cluster 7, Ϫ193; cluster 8, Ϫ190; cluster 9, Ϫ187; cluster 10, Ϫ184; cluster 11, Ϫ181; cluster 12, Ϫ178; cluster 13, Ϫ175; cluster 14, Ϫ172; cluster 15 produced a supershift of the DNA-protein complexes, whether the labeled probe contained ADE5,7 sequences (Fig. 3B, lanes 3-5) or ARS1 sequences (data not shown).…”

“…Plasmid pCB841 (2), which carries BAS2, was partially digested with EcoRI to generate a 2.5-kb fragment that was ligated into the EcoRI site of pR174 to yield pR175. Plasmid M2025 carrying the (His) 6 -BAS2 fusion was a gift of D. Stillman (15).…”

“…Electrophoretic Mobility Shift Assays of DNA Binding by BAS2-GST-BAS2 was prepared and used in EMSAs as described above for GST-BAS1. The (His) 6 -BAS2 protein was prepared for use in EMSA experiments as described (13,15) with the following modification. Crude cell lysates (1.5 mg) from bacteria transformed with GST-BAS2 plasmid pM2025 (13) or the empty vector were prepared and added to 200 l of Ni 2ϩ charged His-Bind resin (Novagen).…”

“…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).…”