Transcription complex formation at the mouse rDNA promoter involves the stepwise association of four transcription factors and RNA polymerase I.
We have used purified transcription factors and RNA polymerase I (pol I) to analyze the individual steps involved in the formation of transcription initiation complexes at the mouse ribosomal gene promoter in vitro. Complete assembly of transcription complexes requires pol I and at least four auxiliary factors, termed TIF-IA, TIF-IB, TIF-IC, and UBF. Preincubation and template commitment, as well as order of addition protocols, were used to discriminate between various intermediate complexes generated during assembly of the initiation complex. As a first step, TIF-IB binds to the core promoter, a process that is facilitated by the upstream control element and the upstream binding factor (UBF). Binding of TIF-IB to the rDNA promoter results in the formation of a functional preinitiation complex (complex 1), which is stable for many rounds of transcription. UBF, which on its own does not stably associate with the rDNA promoter, triggers a 5-10-fold increase in the overall amount of this primary complex. Following binding of TIF-IB and UBF to the template DNA, pol I and TIF-IC successively bind, yielding complexes 2 and 3, respectively. Transcription-competent initiation complexes are built up by the final association of the growth-regulated factor TIF-IA. The various complexes can be distinguished by their different sensitivity to Sarkosyl. Only the complete complex consisting of all four factors and pol I shows resistance to intermediate concentrations of Sarkosyl (0.045%) and is competent to catalyze the formation of the first phosphodiester bond. The initiated complex is, on the other hand, resistant to high concentrations of Sarkosyl (0.3%). The hierarchical nature of the different complexes formed suggests a model for transcription initiation and predicts functions for the individual factors.
Schnapp
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Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Grummt
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D000818:Animals; D002853:Chromatography, Liquid; D004275:DNA, Ribosomal / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism*; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004275:DNA, Ribosomal; D014157:Transcription Factors; D012318:RNA Polymerase I