Nucleic Acids Research, 2000, Vol. 28, No. 6 1390-1396
© 2000 Oxford University Press
Different roles for Abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene
Department of Biochemistry and Molecular Biology, IMBW, Biocentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands and 1Section of Molecular Biology, Swammerdam Institute for Life Science, Universiteit van Amsterdam, Amsterdam, The Netherlands
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 DNaseI digestion showed that the RPS28A promoter contains a 50–60 bp long nucleosome-free region directly downstream from the Abf1p binding site, followed by an ordered array of nucleosomes. Mutating either the Abf1p binding site or the T-rich element has dramatic, but different, effects on the local chromatin structure. Failure to bind Abf1p appears to cause nucleosome positioning to become disorganized as concluded from the complete disappearance of Mnase hypersensitive sites. On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by ~50 bp towards the Abf1p site, resulting in loss of the nucleosome-free region downstream of Abf1p. We conclude that Abf1p is a strong organizer of local chromatin structure that appears to act as a nucleosomal boundary factor requiring the downstream T-rich element to create a nucleosome-free region.
* To whom correspondence should be addressed. Tel: +31 20 4447569; Fax: +31 20 4447553; Email: mager@chem.vu.nl
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