RNA polymerase I holoenzyme-promoter interactions.
In plants and animals, RNA polymerase I (pol I) can be purified in a form that is self-sufficient for accurate rRNA gene promoter-dependent transcription 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 revealed protection of the core promoter region from approximately -30 to +20, in good agreement with the boundaries of the minimal promoter defined by deletion analyses (-33 to +6). Using conventional polyacrylamide electrophoretic mobility shift assays (EMSA), protein-DNA complexes were mostly excluded from the gel. However, agarose EMSA revealed promoter-specific binding activity that co-purified with promoter-dependent transcription activity. Titration, time-course, and competition experiments revealed the formation or dissociation of a single protein-DNA complex. This protein-DNA complex could be labeled by incorporation of radioactive ribonucleotides into RNA in the presence of alpha-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 single DNA binding event.
Saez-Vasquez
J
J
Biology Department, Washington University, St. Louis, Missouri 63130, USA.
Pikaard
C S
CS
D001937:Brassica / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D016172:DNA Fingerprinting; D003851:Deoxyribonucleases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics* / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics; D014158:Transcription, Genetic
D012335:RNA, Ribosomal; D004247:DNA; D012318:RNA Polymerase I; D003851:Deoxyribonucleases