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. 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 MET16UAS 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.
Kuras
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L
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Barbey
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Thomas
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D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D016350:Leucine Zippers / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D015342:DNA Probes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C099899:MET28 protein, S cerevisiae; C073675:MET4 protein, S cerevisiae; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I