No 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.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 ...

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II). This change is reversible and does not involve a simple mutation. The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis. Thus, S. cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.