Binding specificity of the G1/S transcriptional regulators in budding yeast

• Published in: PloS one 2013-04, Vol.8 (4), p.e61059
• Main Authors: Harris, Michael R, Lee, Dave, Farmer, Sarah, Lowndes, Noel F, de Bruin, Robertus A M
• Format: Article
• Language: English
• Subjects: Antisera; Baking yeast; Binding; Binding sites; Biology; Cell cycle; Cell division; Cyclins - genetics; Deoxyribonucleic acid; DNA; DNA-Binding Proteins - metabolism; Feedback loops; G1 Phase; Gene expression; Gene Expression Regulation, Fungal; Gene regulation; Genetic aspects; Genomes; Immunoglobulins; Kinases; Laboratories; Phosphatase; Phosphorylation; Physiological aspects; Positive feedback; Promoter Regions, Genetic; Protein Binding; Protein Interaction Mapping; Proteins; Repressor Proteins - metabolism; Ribonucleotide Reductases - genetics; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Transcription (Genetics); Transcription factors; Transcription Factors - metabolism; Transcription, Genetic; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0061059

G1/S transcriptional regulation in the budding yeast Saccharomyces cerevisiae depends on three main transcriptional components, Swi4, Swi6 and Mbp1. These proteins constitute two transcription factor complexes that regulate over 300 G1/S transcripts, namely SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). SBF and MBF are involved in regulating largely non-overlapping sets of G1/S genes via clearly distinct mechanisms. Here we establish and confirm protein-protein and protein-DNA interactions using specific polyclonal antisera to whole Swi6 and to the C-terminal domains of related proteins Swi4 and Mbp1. Our data confirm the protein-protein binding specificity of Swi4 and Mbp1 to Swi6 but not to each other, and support the binding specificity of the transcriptional inhibitor Whi5 to SBF and of the corepressor Nrm1 to MBF. We also show the DNA binding preference of Swi4 to the CLN2 promoter and Mbp1 to the RNR1 promoter, while Swi6 binds both promoters. Finally, we establish the binding dynamics of Swi4 and Whi5 to the CLN2 promoter during the cell cycle. These data confirm the binding specificity of the G1/S transcriptional regulators. Whereas previous observations were made using tagged Swi4, Swi6 and Mbp1, here we use specific polyclonal antisera to reestablish the protein-protein and protein-DNA interactions of these G1/S transcriptional components. Our data also reveal the dynamic changes in promoter binding of Swi4 during the cell cycle, which suggests a possible positive feedback loop involving Swi4.