title: Distinct DNA elements contribute to Rap1p affinity for its binding sites.
abstract: The essential Saccharomyces cerevisiae regulatory protein Rap1 contains two tandem Myb-like DNA binding sub-domains that interact with two defined DNA "hemisites", separated by a trinucleotide linker sequence. We have mapped the thermodynamically defined DNA-binding site of Rap1 by a primer extension method coupled with electrophoretic separation of bound and unbound DNAs. Relative to published consensus sequences, we detect binding interactions that extend 3 bp beyond the 5'-end of the putative DNA-binding site. This new site of interaction is located where the DNA minor groove faces the protein, and may account for the major DNA bending induced by Rap1p that previous studies have mapped to a site immediately upstream of the consensus binding site. In addition, we show that a minimal DNA-binding site made of one single consensus hemisite, preceded or followed by a spacer trinucleotide that interacts with the unstructured protein linker between the two Rap1p DNA binding domains, is able to bind the protein, although at lower affinity. These findings may explain the observed in vivo binding properties of Rap1p at many promoters that lack canonical binding sites.
authors.affiliation: Dipartimento di Biologia Cellulare e dello Sviluppo, Università La Sapienza di Roma, Rome, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: Del Vescovo
authors.forename: Veronica
authors.initials: V
authors.lastname: De Sanctis
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bianchi
authors.forename: David
authors.initials: D
authors.lastname: Shore
authors.forename: Ernesto
authors.initials: E
authors.lastname: Di Mauro
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Negri
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002843:Chromatin; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors; D004247:DNA