A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports of human SL1. UBF itself does not bind stably to rDNA but rapidly associates and dissociates. We show that SL1 significantly reduces the rate of dissociation of UBF from the rDNA promoter. Our findings challenge the idea that UBF activates transcription through recruitment of SL1 at the rDNA promoter and suggest that the rate of pre-initiation complex (PIC) formation is primarily determined by the rate of association of SL1, rather than UBF, with the promoter. Therefore, we propose that SL1 directs PIC formation, functioning in core promoter binding, RNA polymerase I recruitment, and UBF stabilization and that SL1-promoter complex formation is a necessary prerequisite to the assembly of functional and stable PICs that include the UBF activator in mammalian cells.RNA polymerase (Pol) 1 enzymes themselves have no intrinsic ability to recognize and bind specifically to promoter DNA sequences, so pre-initiation complex formation in transcription calls for the recruitment of the Pol enzymes to the promoter via transcription factors. Basal transcription factors and (co-)activators of transcription cooperate in this function in eukaryotes where three classes of highly related enzymes, Pol I, Pol II, and Pol III, catalyze the transcription of specific sets of genes. With few exceptions, a complex of TBP and TBP-associated factor (TAF) proteins is required for the accurate initiation of transcription by all three polymerases (1). The particular complement of TAFs in each complex, although variable in Pol II transcription, is specific to each class of genes. There is evidence that binding of certain TAF subunits to TBP precludes the binding of TAFs from a different class (2). The precise roles of the TBP-TAF complexes in mediating a specific interaction between the polymerases and their respective promoters are distinct.In mammalian Pol II transcription, the TBP-TAF complex TFIID can bind at the promoter by virtue of the specific interaction of TBP with TATA boxes and the specific interactions of the TAF proteins with other promoter sequences, whereupon Pol II and other factors are recruited to form the pre-initiation complex (PIC) (3). The initial phases of mammalian Pol III transcription from the different types of Pol III promoters converge on the recruitment of the TBP-TAF complex TFIIIB and the Pol III enzyme in formation of the PIC (4, 5). Loading of promoter type-specific TFIIIB at the promoter DNA occurs with the assistance of the specific DNA binding capabilities of TFIIIA and TFIIIC or TFIIIC alone or the multisubunit compl...

Ribosomal RNA gene transcription by RNA polymerase I (Pol I) is the driving force behind ribosome biogenesis, vital to cell growth and proliferation. The key activator of Pol I transcription, UBF, has been proposed to act by facilitating recruitment of Pol I and essential basal factor SL1 to rDNA promoters. However, we found no evidence that UBF could stimulate recruitment or stabilization of the pre-initiation complex (PIC) in reconstituted transcription assays. In this, UBF is fundamentally different from archetypal activators of transcription. Our data imply that UBF exerts its stimulatory effect on RNA synthesis, after PIC formation, promoter opening and first phosphodiester bond formation and before elongation. We provide evidence to suggest that UBF activates transcription in the transition between initiation and elongation, at promoter escape by Pol I. This novel role for UBF in promoter escape would allow control of rRNA synthesis at active rDNA repeats, independent of and complementary to the promoter-specific targeting of SL1 and Pol I during PIC assembly. We posit that stimulation of promoter escape could be a general mechanism of activator function.