DNA-binding requirements of the yeast protein Rap1p as selected in sillico from ribosomal protein gene promoter sequences

Motivation: High-level transcriptional activation of most ribosomal protein (rp) genes in Saccharomyces cervisiae is promoted by the global DNA-binding factor Rap1p. The creation of the complete database of yeast rp gene promoter sequences enabled us to develop a computational selection strategy aimed at acquiring detailed information concerning the DNA-binding specificity of Rap1p. Results: Rap1p sites in rp gene promoters are often found in duplicate, exhibiting strong preferences in both spacing and orientation. Using these preferences, a weight matrix was selected that represents the in vivo binding requirements of Rap1p. The resulting matrix renders the identification of functional Rap1p binding sites more accurate and allowed us to re-evaluate previous in vitro data. Tandemly arranged Rap1p binding sites appear to be typical for rp gene promoters and differ in preferred spacing from sites occurring in (sub)telomeric repeats. The preferred spacing that is found in duplicate Rap1p binding sites of rp gene promoters is restricted to a small window between 15 and 26 bp. This is proposed to reflect the borders within which binding co-operativity operates. The data presented clearly illustrate that computational selection strategies provide information that reaches beyond experimental data. Availability: The rp database is available at the url: http://www.chem.vu.nl/BMB/Database.html

Different roles for Abf1p and a T-rich promotor 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 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