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.Of the three nuclear RNA polymerases, it is RNA polymerase I (pol I) 1 that synthesizes large rRNAs. In Saccharomyces cerevisiae, a precursor 35 S rRNA is transcribed and then processed into the mature 18 S, 5.8 S, and 25 S rRNAs found in ribosomes. These rRNAs are encoded by 100 -200 direct rDNA repeats on chromosome XII. Each spacer region between the pol I-driven 35 S transcription units contains a gene encoding the remaining rRNA, 5 S rRNA, transcribed by pol III.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). Such rrn mutants were expected to be defective in pol I activity in vivo, and confirming this, mutations in genes encoding subunits of pol I (those not shared with either pol II or pol III) were isolated (2). Other mutations that also caused defects in rRNA synthesis (as assessed by pulse labeling in vivo) eventually proved to lie in genes encoding (subunits of) pol I transcription factors.An important advance in the study of yeast pol I was the development of an in vitro transcription system using a crude extract (3-5) and, later, fractionated extracts (6 -8). Extracts from rrn mutant strains were not active, and their activity could be restored by addition of fractions from a wild-type extract. This was used as an assay for the purification of pol I transcription factors (6). 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).Like higher eukaryotes, the yeast pol I promoter is composed of a core element that is essential for transcription, located roughly between ϩ5 and Ϫ40 relative to the start site ...