Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding.
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.
McNabb
D S
DS
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Xing
Y
Y
Guarente
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins; D005800:Genes, Fungal / Q000235:genetics*; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D014157:Transcription Factors
D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors