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Eukaryotic Cell, November 2005, p. 1829-1839, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1829-1839.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA Complex in Saccharomyces cerevisiae

David S. McNabb* and Inés Pinto

Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701

Received 30 July 2005/ Accepted 25 August 2005

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 hap4{Delta} 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.

* Corresponding author. Mailing address: Department of Biological Sciences, SCEN601, University of Arkansas, Fayetteville, AR 72701. Phone: (479) 575-3797. Fax: (479) 575-4010. E-mail: dmcnabb{at}uark.edu.

Eukaryotic Cell, November 2005, p. 1829-1839, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1829-1839.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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