CLONING OF YEAST HAP5 - A NOVEL SUBUNIT OF A HETEROTRIMERIC COMPLEX REQUIRED FOR CCAAT BINDING

The CCAAT-binding factor is a conserved heteromeric transcription fact or 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 thes e genes in response to growth in a nonfermentable carbon source. Previ ous studies have demonstrated that the HAP2, HAP3, and HAP4 subunits o f the yeast CCAAT-binding factor are required for the transcriptional activation of genes containing a CCAAT box. Using the two-hybrid scree ning method, we have identified an additional component of the CCAAT-b inding factor. We present the identification and characterization of a novel gene, HAP5, that encodes an additional subunit of the CCAAT-bin ding factor required for the assembly and DNA-binding activity of the complex. In a hap5 mutant, we show that CCAAT-binding activity is abol ished in vitro. Furthermore, we demonstrate that purified recombinant HAP2, HAP3, and HAP5 are able to reconstitute CCAAT-binding activity i n mobility shift analysis. These data suggest that the HAP2/3/5 hetero trimer represents a unique DNA-binding factor in which all three subun its of the complex are absolutely required for DNA-binding activity.