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.
Mesh Terms:
Amino Acid Sequence, CCAAT-Binding Factor, Carbon, Culture Media, DNA, DNA-Binding Proteins, Fungal Proteins, Gene Expression Regulation, Fungal, Genetic Complementation Test, Macromolecular Substances, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Protein Subunits, RNA, Messenger, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Trans-Activators, Transcription Factors
Amino Acid Sequence, CCAAT-Binding Factor, Carbon, Culture Media, DNA, DNA-Binding Proteins, Fungal Proteins, Gene Expression Regulation, Fungal, Genetic Complementation Test, Macromolecular Substances, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Protein Subunits, RNA, Messenger, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Trans-Activators, Transcription Factors
Eukaryotic Cell
Date: Nov. 01, 2005
PubMed ID: 16278450
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