In Vivo Topography of Rap1p–DNA Complex at Saccharomyces cerevisiae TEF2 UASRPG During Transcriptional Regulation
Copyright policy )In Vivo Topography of Rap1p–DNA Complex at Saccharomyces cerevisiae TEF2 UASRPG During Transcriptional Regulation
We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed.
- Roma Tre University Italy
- University of Trento Italy
- Sapienza University of Rome Italy
- University of Geneva Switzerland
- University of Cagliari Italy
Models, Molecular, 570, Saccharomyces cerevisiae Proteins, Base Sequence, Transcription, Genetic, Macromolecular Substances, Molecular Sequence Data, DNA Footprinting, rap1 GTP-Binding Proteins, Saccharomyces cerevisiae, Peptide Elongation Factors, Chromatin, DNA-Binding Proteins, Kinetics, Peptide Elongation Factor 1, Micrococcal Nuclease, Amino acid starvation; Chromatin; Potassium permanganate; Rap1p; Transcription regulation; Amino Acids; Base Sequence; Chromatin; DNA Footprinting; DNA, Fungal; DNA-Binding Proteins; Kinetics; Macromolecular Substances; Micrococcal Nuclease; Models, Molecular; Molecular Sequence Data; Peptide Elongation Factors; Promoter Regions, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription, Genetic; rap1 GTP-Binding Proteins; Virology, Amino Acids, DNA, Fungal, Promoter Regions, Genetic
Models, Molecular, 570, Saccharomyces cerevisiae Proteins, Base Sequence, Transcription, Genetic, Macromolecular Substances, Molecular Sequence Data, DNA Footprinting, rap1 GTP-Binding Proteins, Saccharomyces cerevisiae, Peptide Elongation Factors, Chromatin, DNA-Binding Proteins, Kinetics, Peptide Elongation Factor 1, Micrococcal Nuclease, Amino acid starvation; Chromatin; Potassium permanganate; Rap1p; Transcription regulation; Amino Acids; Base Sequence; Chromatin; DNA Footprinting; DNA, Fungal; DNA-Binding Proteins; Kinetics; Macromolecular Substances; Micrococcal Nuclease; Models, Molecular; Molecular Sequence Data; Peptide Elongation Factors; Promoter Regions, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription, Genetic; rap1 GTP-Binding Proteins; Virology, Amino Acids, DNA, Fungal, Promoter Regions, Genetic
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