Genome-wide regulation of TATA-binding protein activity

Abstract

Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different protein complexes, which can regulate the activity of TBP positively as well negatively. In order to understand how TBP activity is controlled, the binding of TBP and TBP-containing complexes was mapped across the genome in a model organism, Saccharomyces cerevisiae. To achieve this, a technique called chromatin immunoprecipitation in combination with microarray technology (ChIP-chip) was used. The steps of the ChIP-chip procedure involve crosslinking of protein-DNA interaction, sonication into small DNA fragments, purification of protein-DNA complexes, amplification of ChIP material, and hybridization to microarrays. In order to get high quality and high resolution ChIP-chip datasets, the ChIP procedure was improved at several steps. First, to purify the protein-DNA complexes with great efficiency, a biotinylation tagging approach was used. Biotin interacts with streptavidin with extreme high affinity, and therefore allows stringent washing conditions in the ChIP procedure. Second, a T7- RNA polymerase-based linear amplification procedure was adapted to amplify ChIP material to sufficient amounts for hybridization to microarrays. This method performed better compared to traditionally used PCR amplification methods. Using these optimized steps of the ChIP-chip procedure, we measured promoter occupancy by TFIID/SAGA (positive regulators of TBP) and NC2/Mot1p (negative regulators of TBP). Strikingly, NC2, TBP, and Mot1p binding overlap at a substantial number of promoters. Interestingly, many of these promoters are also occupied by the positive regulators of TBP, TFIID and SAGA. Affinity-purification of Mot1p revealed that NC2, Mot1p, and TBP form a stable complex on chromatin, which is dynamically regulated by the ATP hydrolysis via Mot1p. These results lead to a model, in which dynamic exchange between positive and negative regulators is essential for regulation TBP activity and gene transcription.