Different roles for Abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene

In vivo mutational analysis of the yeast RPS28A ribosomal protein (rp-)gene promoter demonstrated that both the Abf1p binding site and the adjacent T- rich element are essential for efficient transcription, In vivo Mnase and D Nasel digestion showed that the RPS28A promoter contains a 50-60 bp long nu cleosome-free region directly downstream from the Abf1p binding site, follo wed by an ordered array of nucleosomes. Mutating either the Abf1p binding s ite or the T-rich element has dramatic, but different, effects on the local chromatin structure. Failure to bind Abf1p appears to cause nucleosome pos itioning to become disorganized as concluded from the complete disappearanc e of Mnase hypersensitive sites. On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by similar to 50 b p towards the Abf1p site, resulting in loss of the nucleosome-free region d ownstream of Abf1p, We conclude that Abf1p is a strong organizer of local c hromatin structure that appears to act as a nucleosomal boundary factor req uiring the downstream T-rich element to create a nucleosome-free region.