RNA polymerase I holoenzyme-promoter interactions

In plants and animals, RNA polymerase I (pol I) can be purified in a form t hat is self-sufficient for accurate rRNA gene promoter-dependent transcript ion and that has biochemical properties suggestive of a single complex, or holoenzyme, In this study, we examined the promoter binding properties of a highly purified Brassica pol I holoenzyme activity. DNase I footprinting r evealed protection of the core promoter region from similar to -30 to +20, in good agreement with the boundaries of the minimal promoter defined by de letion analyses (-33 to +6), Using conventional polyacrylamide electrophore tic mobility shift assays (EMSA), protein-DNA complexes were mostly exclude d from the gel. However, agarose EMSA revealed promoter-specific binding ac tivity that co purified with promoter-dependent transcription activity. Tit ration, time-course, and competition experiments revealed the formation or dissociation of a single protein-DNA complex, This protein-DNA complex coul d be labeled by incorporation of radioactive ribonucleotides into RNA in th e presence of rw-amanitin, suggesting that the polymerase I enzyme is part of the complex. Collectively, these results suggest that transcriptionally competent pol I holoenzymes can associate with rRNA gene promoters in a sin gle DNA binding event.