In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) 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.
De Sanctis
Veronica
V
Fondazione "Istituto Pasteur-Fondazione Cenci-Bolognetti", c/o Dipartimento di Genetica e Biologia Molecolare, Università di Roma, La Sapienza, Rome, Italy.
La Terra
Sabrina
S
Bianchi
Alessandro
A
Shore
David
D
Burderi
Luciano
L
Di Mauro
Ernesto
E
Negri
Rodolfo
R
D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; C057183:TEF2 protein, S cerevisiae; D008836:Micrococcal Nuclease; D020827:rap1 GTP-Binding Proteins