Evidence that partial unwrapping of DNA from nucleosomes facilitates the binding of heat shock factor following DNA replication in yeast.
In the yeast Saccharomyces cerevisiae, heat shock transcription factor (HSF) binds heat shock element (HSE) DNA shortly after DNA replication, independently of its activation by heat shock. To determine if HSF binding occurs before newly replicated DNA is packaged into nucleosomes, we inserted an HSE into a DNA segment that normally forms a positioned nucleosome in vivo. Transcription from constructs designed to create steric competition between binding of HSF and histone H2A-H2B dimers was generally poor, suggesting that nucleosome assembly precedes and inhibits HSF binding. However, one such construct was as transcriptionally active as a nucleosome-free control. Structural analyses suggested that approximately 40 base pairs of DNA, including the HSE, had unwrapped from the 3' edge of the histone octamer, allowing HSF to bind; approximately 100 base pairs remained in association with the histone octamer, with the same translational and rotational orientation as was seen for the poorly transcribed constructs. Modeling studies suggest that the active and inactive constructs differ from one another in the ease with which the HSE and flanking sequences can adopt the curvature needed to form a stable nucleosome. These differences may influence the probability of DNA unwrapping from already assembled nucleosomes and the subsequent binding of HSF.
Geraghty
D S
DS
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405-0068, USA.
Sucic
H B
HB
Chen
J
J
Pederson
D S
DS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004261:DNA Replication / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D006657:Histones; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease