Reduction of ribonucleotides into deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase. The mouse enzyme is composed of two protein subunits, the R1 protein and the R2/p53R2 protein, and its subunit constellation differs during the cell cycle. We report here how the promoters of each of these subunits are regulated during the cell cycle. Previous DNase footprinting experiments of the R1 and the R2 promoter gave us an idea of how these promoters are structured. The R1 promoter contains four elements; Inr, α (binding YY1), β (binding YY1) and γ, while the R2 promoter contains four different elements; TATA-box (binding TBP), CCAAT-box (binding NFY), E2F element (binding E2F4) and an upstream activating region. The p53R2 promoter is uncharacterized; only the transcription start has been suggested in Genebank.
We found that activation of both subunits needed for S phase specific activity (R1 and R2) is dependent of release of the repressor E2F4 from each promoter. Previous results showed that the mouse R2 promoter harbors an E2F4 binding element and our result, using transient transfections, indicates that this is also the case for the mouse R1 promoter. Using primer extension experiments on the mouse p53R2 promoter we show that the transcription start colocalizes with an earlier unidentified Inr element similar to the Inr element in the mouse R1 promoter. Our transcription start site is localized 126 bp downstream from the start site indicated in Genebank. We also show that it is possible to partially purify the transcription factor(s) binding to the upstream activating region in the mouse R2 promoter by using phosphocellulose chromatography and oligonucleotides immobilized on magnetic beads.