Reconstitution of Transcription Factor SL1: Exclusive Binding of TBP by SL1 or TFIID Subunits

Abstract

RNA polymerase I and II transcription factors SL1 and TFIID, respectively, are composed of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) responsible for promoter recognition. How the universal transcription factor TBP becomes committed to a TFIID or SL1 complex has not been known. Complementary DNAs encoding each of the three TAFIs that are integral components of SL1 have not been isolated. Analysis of subunit interactions indicated that the three TAFIs can bind individually and specifically to TBP. In addition, these TAFIs interact with each other to form a stable TBP-TAF complex. When TBP was bound first by either TAFI110, 63, or 48, subunits of TFIID such as TAFII250 and 150 did not bind TBP. Conversely, if TBP first formed a complex with TAFII250 or 150, the subunits of SL1 did not bind TBP. These results suggest that a mutually exclusive binding specificity for TBP intrinsic to SL1 and TFIID subunits directs the formation of promoter- and RNA polymerase-selective TBP-TAF complexes.

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

“…hTAF I 63 has a 40-amino acid extension at its N terminus that the mouse protein lacks whereas mTAF I 68 contains an additional 66 amino acids at the C terminus. The two putative zinc finger motifs present in human TAF I 63 (12) are conserved in the murine protein, and the C-terminal extension in mTAF I 68 allows for the possibility of a third zinc finger (amino acids 486-490 and 518-522). This additional zinc finger in mTAF I 68 could explain why TIF-IB can form a stable, committed complex in the absence of UBF and SL1 cannot (3,4).…”

“…Two cDNA clones (2.4 and 1.56 kb) containing an identical 1.4 kb ORF were isolated. cDNAs encoding mTAF I 68 and mTAF I 95 were isolated from mouse cDNA libraries using DNA fragments derived from the respective human TAF I cDNA (12). The full length ORFs encoding mTAF I 68 and mTAF I 95 were reconstructed by fusion of two partial cDNAs.…”

“…GST-mTBP ''pull-down'' assays were performed as described (12). For TAF-TAF interaction studies, M2 antibody beads (Kodak) were incubated with extracts from Sf9 cells containing FLAG-tagged mTAF I s at 4ЊC in buffer TM-400 (400 mM KCl͞50 mM Tris⅐HCl, pH 7.9͞12.5 mM MgCl 2 ͞10% glycerol͞1 mM DTT͞0.2 mM PMSF͞1 mM sodium metabisulfide͞0.1% Nonidet P-40).…”

“…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 encoding the mouse pol I-specific TBP-associated factors (TAF I s) subunits.…”

Breaking the mold: structures of the RNA polymerase I transcription complex reveal a new path for initiation

“…It is also clear from in vitro studies that TBP stimulates yeast Pol I transcription activity [29], indicating that TBP acts more like a coactivator in the Pol I system rather than a basal initiation factor. Given that TBP is a stable component of human Selectivity Factor 1 (SL1) unlike CF [30,31], this also leaves open the possibility that TBP may be utilized differently between yeast and human Pol I systems.…”