TFIIA Regulates TBP and TFIID Dimers Robert A Coleman, Andrew K.P Taggart, Sandeep Burma, John J Chicca, B.Franklin Pugh  Molecular Cell  Volume 4, Issue 3, Pages 451-457 (September 1999) DOI: 10.1016/S1097-2765(00)80453-0

Figure 1 Recombinant TFIIA Promotes the Dissociation of TBP Dimers Recombinant human ([A], 50 nM) or yeast ([B], 125 nM) wild-type or TFIIA-defective mutant TBPs, as indicated, were incubated with the indicated concentration of recombinant human or yeast TFIIA, respectively. After 60 min at 25°C, reactions were briefly treated with BMH and analyzed by Western blotting. (C) Recombinant wild-type hTBP (1 μM) was incubated for 30 min at 0°C with resin lacking (lane 1) or containing GST-hTBP(180C) (1 μM, lanes 2–4). Resins were washed extensively, then incubated with recombinant hTFIIA (1.25 μM, lane 3) or TATA DNA (40 μM, lane 4). Incubations at 25°C were continued for 60 min. Resins were washed again, and resin-bound hTBP was analyzed by Western blotting. (D) Recombinant hTBP (250 nM) was incubated in the absence or presence of recombinant hTFIIA (1.5 μM) for 60 min at 0°C. DNA binding was initiated by diluting the reaction 25-fold (to 10 nM TBP and 60 nM TFIIA) into buffer containing 25 nM [32P] TATA DNA to initiate the binding reaction. TBP/TATA association was measured using a filter binding assay (Coleman and Pugh 1997). Similar rate accelerations were obtained when binding was initiated without preincubation of TFIIA and TBP, but due to the relatively low bimolecular association rate constant for the interaction of TFIIA and DNA-free TBP, higher TFIIA concentrations were required (data not shown). The rate acceleration in the absence of a preincubation indicates that TFIIA induces TBP dimer dissociation instead of trapping monomers as dimers dissociate at their intrinsic rate. Molecular Cell 1999 4, 451-457DOI: (10.1016/S1097-2765(00)80453-0)

Figure 2 TFIID Dimers Are Slow to Dissociate (A) A schematic of the pulldown assay used to measure the rate of TFIID dimer dissociation. TFIID (containing ∼0.1 nM TBP) from a phosphocellulose P.7 fraction (B and C) or a more purified Mono Q fraction (D) was mixed with his-180C resin (200 nM) in 500 μl. Where indicated, control reactions lacked his-180C. At the indicated time, the resin was collected by centrifugation, and the TBP present in the supernatant (after TCA precipitation) and resin pellet determined by Western blotting with hTBP (B and D) or TAFII250 (C) antibodies. The TBP band intensities from four experiments were quantitated and plotted as a function of time, and fit to a single exponential. A small lag of 3–5 min was reproducibly observed and was excluded from the curve fitting. Additional controls, not shown, included the use of a resin-bound his-180C containing a dimerization-defective mutation (Jackson-Fisher et al. 1999a), which did not retain TFIID; inclusion of excess untagged wild-type yTBP, which readily bound his-180C and inhibited the pulldown of TFIID; and the use of up to 3-fold higher his-180C concentrations, which did not alter the kinetics. These controls indicated that the pulldown is specific and not due to a general aggregation of TFIID and that the observed reaction rate is intrinsic to TFIID and not the his-180C resin. Other details and controls are described elsewhere (Jackson-Fisher et al. 1999a). We have also examined the exchange of TAFII250 from endogenous TFIID onto immobilized TBP and found very little stable exchange under these conditions over a period of ∼24 hr (Pugh and Tjian 1991). Molecular Cell 1999 4, 451-457DOI: (10.1016/S1097-2765(00)80453-0)

Figure 3 Slow Loss of TFIID Dimers in the Presence of TATA-Containing Promoter DNA (A) G6I or G6TI promoters were titrated into reactions at the indicated concentration. The total DNA concentration was kept constant with poly dG-dC. After 80 min of incubation at 23°C, reactions were cross-linked for 90 s with 1 mM BMH, and hTBP was analyzed by Western blotting. XL dimer denotes cross-linked dimer, and Un-XL denotes un-cross-linked TBP. (B) Reactions are the same as in lane 8 of (A), except that the reactions were subjected to BMH cross-linking at the indicated time points. The data are plotted in Figure 4E. Molecular Cell 1999 4, 451-457DOI: (10.1016/S1097-2765(00)80453-0)

Figure 4 TFIIA Induces TFIID Dimer Dissociation (A) One nanomolar TFIID (P.7) was incubated in the absence of DNA and in the presence or absence of 10 nM TFIIA. Cross-linking reactions were performed as described in Figure 3. (B) Reactions are the same as in Figure 3, using the indicated concentrations of TFIIA, G6I, G6TI, and TFIID. The asterisk denotes a spurious irrelevant band. (C) Reactions are as in (B) but contained 0.4 nM TFIID, 30 nM G5E4T promoter fragment, and either no TFIIA (lane 1) or TFIIA immunodepleted with equal amounts of nonspecific (lane 2) or TFIIA (lane 3) antibodies. (D) Reactions are the same as in lane 11 of (B), except that the reactions were subjected to BMH cross-linking at the indicated time points. (E) The band intensities of the XL dimer in (D) were quantitated and plotted as a function of time and compared to the data in Figure 3B. Molecular Cell 1999 4, 451-457DOI: (10.1016/S1097-2765(00)80453-0)

Figure 5 A Model for How TFIIA Might Enhance the Loading of TFIID onto Promoter DNA A slow dissociation of TFIID dimers presents a formidable kinetic barrier to promoter binding. TFIIA binds TFIID weakly and induces dimer dissociation. If promoter DNA is nearby, it traps and stabilizes TFIID in a monomeric and transcriptionally active state. If promoter DNA is not nearby, the unstable TFIIA/TFIID complex collapses back to the more stable TFIID dimer and free TFIIA. Molecular Cell 1999 4, 451-457DOI: (10.1016/S1097-2765(00)80453-0)