title: The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes.
abstract: Budding yeast Rap1 is a specific double-stranded DNA-binding protein involved in repression and activation of gene transcription and in the establishment of the nucleoprotein complex formed at telomeres. The DNA-binding domain (DBD) of Rap1 forms a high affinity complex with DNA where both Myb-like domains bind to the recognition site. However, we recently showed that the DBD can also access an alternative, lower affinity DNA-binding mode where a single Myb-like domain binds. This results in Rap1-DNA complexes with stoichiometry higher than previously anticipated. In this work, we show that the ability of the DBD to form higher stoichiometry complexes on DNA is maintained also in larger Rap1 constructs. This indicates that transition between at least two DNA-binding modes is a general property of the protein and not a specific feature of the DBD in isolation. The transition between binding modes is modulated by the C-terminal wrapping loop within the DBD, consistent with the proposed model in which the transient opening of this region allows a switch between binding modes. Finally, we provide evidence that the Rap1 C terminus interacts with the DNA-binding domain, suggesting a complex network of interactions that couples changes in conformation of the protein to the binding of its DNA recognition sequence.
authors: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors: Erik A
authors: EA
authors: Feldmann
authors: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors: Paolo
authors: P
authors: De Bona
authors: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110 galletto@biochem.wustl.edu.
authors: Roberto
authors: R
authors: Galletto
mesh_terms: D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors
keywords: Analytical Ultracentrifugation; Binding Modes; Fluorescence Anisotropy; Isothermal Titration Calorimetry (ITC); Rap1; Telomere; Transcription