Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA complex in Saccharomyces cerevisiae.
The CCAAT-binding factor (CBF) is an evolutionarily conserved multimeric transcriptional activator in eukaryotes. In Saccharomyces cerevisiae, the CCAAT-binding factor is composed of four subunits, termed Hap2p, Hap3p, Hap4p, and Hap5p. The Hap2p/Hap3p/Hap5p heterotrimer is the DNA-binding component of the complex that binds to the consensus 5'-CCAAT-3' sequence in the promoter of target genes. The Hap4p subunit contains the transcriptional activation domain necessary for stimulating transcription after interacting with Hap2p/Hap3p/Hap5p. In this report, we demonstrate that Hap2p, Hap3p, and Hap5p assemble via a one-step pathway requiring all three subunits simultaneously, as opposed to the mammalian CCAAT-binding factor which has been shown to assemble via a two-step pathway with CBF-A (Hap3p homolog) and CBF-C (Hap5p homolog) forming a stable dimer before CBF-B (Hap2p homolog) can interact. We have also found that the interaction of Hap4p with Hap2p/Hap3p/Hap5p requires DNA binding as a prerequisite. To further understand the protein-protein and protein-DNA interactions of this transcription factor, we identified the minimal domain of Hap4p necessary for interaction with the Hap2p/Hap3p/Hap5p-DNA complex, and we demonstrate that this domain is sufficient to complement the respiratory deficiency of a hap4Delta mutant and activate transcription when fused with the VP16 activation domain. These studies provide a further understanding of the assembly of the yeast CCAAT-binding factor at target promoters and raise a number of questions concerning the protein-protein and protein-DNA interactions of this multisubunit transcription factor.
McNabb
David S
DS
Department of Biological Sciences, SCEN601, University of Arkansas, Fayetteville, AR 72701, USA. dmcnabb@uark.edu
Pinto
Inés
I
D000595:Amino Acid Sequence; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D023081:CCAAT-Binding Factor; D003470:Culture Media; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C490431:HAP3 protein, S cerevisiae; C061437:HAP4 protein, S cerevisiae; C508483:Hap5 protein, S cerevisiae; D046911:Macromolecular Substances; D021122:Protein Subunits; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C111072:aab-1 protein, Neurospora; D002244:Carbon; D004247:DNA