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 se quence (5'-ACACCCATACATTT-3') and the Saccharomyces cerevisiae telomeric co nsensus (5'-ACACCCACACACCC-3'). When cloned in front of a minimal CYC1 prom oter, the two sequences differed in their transcriptional potential. Wherea s UASrpg or telomeric single binding sites activated transcription with app roximately the same strength, adjacent UASrpg sequences showed higher syner gistic activity and orientation-dependence than telomeric sequences. We als o 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 R ap1p binding domain distorted DNA molecules encompassing the UASrpg sequenc e or the telomeric-like sequence, as revealed by both KMnO4 hypersensitivit y and by hydroxyl radical foot-printing analysis. We propose that Rap1p is able to form structurally and functionally different complexes, depending o n the type of DNA sequence the complex is assembled from. This functional a nd structural heterogeneity may be responsible for the multiple functions t hat Rap1p binding sites appear to have in vivo. (C) 1998 Academic Press.