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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
Author(s) -
Kuras Laurent,
Barbey Régine,
Thomas Dominique
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.9.2441
Subject(s) - biology , transcription factor , yeast , transcription (linguistics) , microbiology and biotechnology , dna , dna binding protein , stimulation , genetics , computational biology , neuroscience , gene , linguistics , philosophy
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. Cbf1 belongs to the basic helix–loop–helix DNA‐binding protein family while Met4 and Met28 are two basic leucine zipper (bZIP) factors. We have shown previously that in cell extracts, the three factors are found in a high molecular weight complex. By using mobility shift assays, we report here that the in vitro reconstitution of the Cbf1–Met4–Met28 complex on MET16 UAS can be obtained with purified recombinant proteins. DNase I protection experiments confirm that the Cbf1–Met4–Met28 complex is formed over the TCACGTG sequence. The experiments also show that both Met4 and Met28 bind to DNA only in the presence of Cbf1. Moreover, Met28 is shown to enhance the DNA‐binding activity of Cbf1. Analysis of MET28 gene regulation reveals that its expression requires Met4. Thus the biochemical activity of Met28 allows the establishment of a positive regulatory loop. The results thus provide evidence of a new functional relationship between bHLH and bZIP proteins and demonstrate that the association of such factors may serve to discriminate between the different TCACGTG sequences found in the chromosomes.