Abstract
Five purified protein components, RNA polymerase I, Rrn3p, core factor, TBP (TATA-binding protein), and upstream activation factor, are sufficient for high level transcription in vitro from the Saccharomyces cerevisiae rDNA promoter. Rrn3p and pol I form a complex in solution that is active in specific initiation. Three protein components, pol I, Rrn3p, and core factor, and promoter sequence to -38, suffice for basal transcription. Unlike pol II and pol III, yeast pol I basal transcription does not require TBP. Instead, TBP, upstream activation factor, and the upstream element of the promoter together stimulate pol I basal transcription to a fully activated level. The role of TBP in pol I transcription is fundamentally different from its role in pol II or pol III transcription.
Highlights
Of the three nuclear RNA polymerases, it is RNA polymerase I1 that synthesizes large rRNAs
(28) had apparently been contaminated with small amounts of core factor (CF) or upstream activation factor (UAF), so polymerase I (pol I) purification was made more stringent. pol I was purified from yeast crude extracts by gradient elution from each of four columns, phosphocellulose, Q Sepharose, heparin-Sepharose, and MonoQ, yielding a preparation that was essentially pure as judged by visual inspection of silverstained gels (Fig. 1), and free of any other pol I factors. pol I that had been Histagged on the second largest subunit (A135) was purified by nickel affinity chromatography followed by gradient elution from heparin and MonoQ resins
Such mutations have a lethal or nearly lethal phenotype, but the phenotype can be rescued by transcription of rRNA from a pol II promoter, indicating that the defect is in pol I transcription [1, 2, 6, 9, 10, 12, 31, 32]
Summary
Of the three nuclear RNA polymerases, it is RNA polymerase I (pol I) that synthesizes large rRNAs. The only essential function of pol I in yeast is synthesis of the 35 S rRNA transcript, since the lethal phenotype of a deletion in the second largest subunit of pol I can be rescued by synthesis of the 35 S rRNA transcript by pol II from a GAL promoter placed correctly upstream of the 35 S transcription unit on a high copy plasmid [1] This provided a screen for mutants dependent on pol II-driven synthesis of rRNA from the GAL promoter [2]. The availability of cloned RRN genes, which could be tagged with the hemagglutinin antigen (HA) or hexahistidine, greatly facilitated purification In this way, the multi-subunit factors, core factor (CF), and upstream activation factor (UAF), and the single subunit factor Rrn3p were identified and shown to be necessary for activity in the crude in vitro system as well as in vivo (6, 9 –12). We demonstrate rigorously that TBP is required for high level activated transcription, it is not required for basal transcription
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