We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAF I 110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAF I 110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAF I complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with UBF. The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.
Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II-and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB͞ SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAF I s) and compared the amino acid sequences of the murine TAF I s with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAF I s, demonstrating that differences in the primary structure of human and mouse TAF I s do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAF I complex is likely to be the result of cumulative subtle differences between individual subunits that lead to speciesspecific properties of RNA polymerase I transcription.Transcription initiation by all three classes of eukaryotic nuclear RNA polymerases is a complex process, requiring concerted interactions between multiple protein factors and RNA polymerase. Each class of RNA polymerase uses a distinct assortment of transcription factors that are thought to nucleate the assembly of transcription initiation complexes at specific promoters. For transcription governed by RNA polymerase I (pol I), the murine transcription initiation factor (TIF) IB and its human homologue SL1 have been shown to direct the assembly of productive initiation complexes at the mouse and human rDNA promoter (1-3). TIF-IB͞SL1 is thought to communicate with the upstream binding factor (UBF) and to recruit pol I together with the associated factors TIF-IA and TIF-IC to the template (4).Earlier studies had revealed that rDNA transcription is speciesspecific, requiring factors from either the same or very closely related species (5). Most of the factors, i.e., UBF, pol I, TIF-IA, and TIF-IC, are interchangeable between human and mouse (3, 6-9) whereas TIF-IB͞SL1 has been found to be the speciesspecific component in the preinitiation complex (3, 9). A significant advance toward a functional characterization of this selectivity factor was the discovery that TIF-IB͞SL1 is a multiprotein complex consisting of TATA box binding protein (TBP) and three TBP-associated factors (TAFs) (10, 11). Given the low abundance of TIF-IB͞SL1 in the cell, studies on the molecular mechanism of promoter recognition and species-specific transcription require the isolation and functional characterization of the individual subunits of TIF-IB and SL1. Recently, this was accomplished for the components of SL1 (12, 13). Here we report the cloning and expression of the cDNAs encodi...
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