Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding

• Published in: Genes & development Vol. 9; no. 1; pp. 47 - 58
• Main Authors: McNabb, D S, Xing, Y, Guarente, L
• Format: Journal Article
• Language: English
• Published: United States 01-01-1995
• Subjects: Amino Acid Sequence; Base Sequence; CCAAT-Binding Factor; CCAAT-Enhancer-Binding Proteins; Cloning, Molecular; DNA, Fungal - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fungal Proteins; Genes, Fungal - genetics; Genome, Fungal; Molecular Sequence Data; Protein Binding; Protein Conformation; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Selection, Genetic; Sequence Analysis, DNA; Transcription Factors
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.9.1.47

The CCAAT-binding factor is a conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites (UASs) within the promoters of numerous eukaryotic genes. The CCAAT-binding factor of Saccharomyces cerevisiae activates the transcription of these genes in response to growth in a nonfermentable carbon source. Previous studies have demonstrated that the HAP2, HAP3, and HAP4 subunits of the yeast CCAAT-binding factor are required for the transcriptional activation of genes containing a CCAAT box. Using the two-hybrid screening method, we have identified an additional component of the CCAAT-binding factor. We present the identification and characterization of a novel gene, HAP5, that encodes an additional subunit of the CCAAT-binding factor required for the assembly and DNA-binding activity of the complex. In a hap5 mutant, we show that CCAAT-binding activity is abolished in vitro. Furthermore, we demonstrate that purified recombinant HAP2, HAP3, and HAP5 are able to reconstitute CCAAT-binding activity in mobility shift analysis. These data suggest that the HAP2/3/5 heterotrimer represents a unique DNA-binding factor in which all three subunits of the complex are absolutely required for DNA-binding activity.