Structural and functional heterogeneity of Rap1p complexes with telomeric and UASrpg-like DNA sequences.
Rap1p binds to a variety of related DNA sequences. We studied complexes of Rap1p and its DNA-binding domain with two of these sequences, the UASrpg sequence (5'-ACACCCATACATTT-3') and the Saccharomyces cerevisiae telomeric consensus (5'-ACACCCACACACCC-3'). When cloned in front of a minimal CYC1 promoter, the two sequences differed in their transcriptional potential. Whereas UASrpg or telomeric single binding sites activated transcription with approximately the same strength, adjacent UASrpg sequences showed higher synergistic activity and orientation-dependence than telomeric sequences. We also found different sequence requirements for Rap1p binding in vitro to both sequences, since a single base-pair that severely reduced binding of Rap1p to UASrpg sequences had very little effect on the telomeric sequence. The Rap1p binding domain distorted DNA molecules encompassing the UASrpg sequence or the telomeric-like sequence, as revealed by both KMnO4 hypersensitivity and by hydroxyl radical foot-printing analysis. We propose that Rap1p is able to form structurally and functionally different complexes, depending on the type of DNA sequence the complex is assembled from. This functional and structural heterogeneity may be responsible for the multiple functions that Rap1p binding sites appear to have in vivo.
Idrissi
F Z
FZ
Centre d'Investigació i Desenvolupament, Consejo Superior de Investigaciones Científicas, Jordi Girona 18, Barcelona, 08034, Spain.
Fernández-Larrea
J B
JB
Piña
B
B
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D017665:Hydroxyl Radical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D034501:Telomere-Binding Proteins; D014157:Transcription Factors; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors; D011196:Potassium Permanganate; D017665:Hydroxyl Radical