ASSEMBLY OF A BZIP-BHLH TRANSCRIPTION ACTIVATION COMPLEX - FORMATION OF THE YEAST CBF1-MET4-MET28 COMPLEX IS REGULATED THROUGH MET28 STIMULATION OF CBF1 DNA-BINDING

Transcriptional activation of sulfur amino acid metabolism in yeast is dependent on a multi-functional factor, the centromere-binding factor 1 (Cbf1) and on two specific transcription factors, Met4 and Met28. C bf1 belongs to the basic helix-loop-helix DNA-binding protein family w hile Met4 and Met28 are two basic leucine zipper (bZIP) factors, We ha ve shown previously that in cell extracts, the three factors are found in a high molecular weight complex. By using mobility shift assays, w e report here that the in vitro reconstitution of the Cbf1-Met4-Met28 complex on MET16(UAS) can be obtained,vith purified recombinant protei ns. DNase I protection experiments confirm that the Cbf1-Met4-Met28 co mplex is formed over the TCACGTG sequence, The experiments also show t hat both Met4 and Met28 bind to DNA only in, the presence of Cbf1. Mor eover, Met28 is shown to enhance the DNA-binding activity of Cbf1. Ana lysis of MET28 gene regulation reveals that its expression requires Me t4. Thus the biochemical activity of Met28 allows the establishment of a positive regulatory loop. The results thus provide evidence of a ne w functional relationship between bHLH and bZIP proteins and demonstra te that the association of such factors may serve to discriminate betw een the different TCACGTG sequences found in the chromosomes.