Cloning of murine RNA polymerase I-specific TAF factors: Conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

Abstract

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...

Mechanism of Repression of RNA Polymerase I Transcription by the Retinoblastoma Protein

“…Western blots of the matrix-bound fraction with anti-TAF I 95 antibodies (14) revealed that the TIF-IB complex was specifically and efficiently retained on the UBF affinity matrix (Fig. 6B, lanes 1 and 2).…”

“…After incubation for 2.5 h at 4°C, the resins were extensively washed with buffer AM-100-0.1% NP-40 and the beads were boiled in SDS-containing buffer and applied to SDS-8% polyacrylamide gels. Pol I and TIF-IB were detected on immunoblots with purified antibodies against RPA116, the second largest subunit of mouse Pol I (37), or antibodies raised against mouse TAF I 95 (14).…”

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

“…Recently, an alternative model was proposed (Grummt, personal communication) based on the finding that mitosis-specific phosphorylation of the hTAF I 110 subunit of SL1 by p34 cdc2 / cyclin B kinase causes repression of rDNA transcription. SL1 is essential for the assembly of preinitiation complexes at the rDNA promoter (Eberhard et al, 1993;Heix et al, 1997) by recruiting RNA pol I associated with two essential factors, TIF-IA and TIF-IC, to the template (Schnapp and Grummt, 1991). Similar mechanisms of transcription repression by mitosisspecific phosphorylation have been found for equivalent TBP-TAF complexes, TFIID of RNA pol II and TFIIIB of RNA pol III, in which assembly of preinitiation complexes is inhibited (Hartl et al, 1993;Gottesfeld et al, 1994;White et al, 1995;Leresche et al, 1996;Segil et al, 1996; reviewed by Gottesfeld and Forbes, 1997).…”

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

“…Speciesspecific gene expression in human and mouse is restricted to some genes involved in, e.g., immune and xenobiotic response, and for polymerase I-dependent transcription of rRNAs (27)(28)(29). In such cases, one could alternatively rescue with a BAC carrying the same gene containing silent mutations or a modified 3Ј UTR.…”