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

Genes Dev. 1995 Jan 1;9(1):47-58. doi: 10.1101/gad.9.1.47.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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 / genetics*
  • Selection, Genetic
  • Sequence Analysis, DNA
  • Transcription Factors

Substances

  • CCAAT-Binding Factor
  • CCAAT-Enhancer-Binding Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors

Associated data

  • GENBANK/U19932