Simian virus 40 large T antigen stabilizes the TATA-binding protein-TFIIA complex on the TATA element

Abstract

Large T antigen (T antigen), the early gene product of simian virus 40 (SV40), is a potent transcriptional activator of both cellular and viral genes. Recently we have shown that T antigen is tightly associated with TFIID and, in this position, performs a TATA-binding protein (TBP)-associated factor (TAF)-like function. Based on this observation, we asked whether T antigen affected steps in preinitiation complex assembly. Using purified components in in vitro complex assembly assays, we found that T antigen specifically enhances the formation of the TBP-TFIIA complex on the TATA element. T antigen accomplishes this by increasing the rate of formation of the TBP-TFIIA complex on the TATA element and by stabilizing the complexes after they are formed on the promoter. In addition, DNA immunoprecipitation experiments indicate that T antigen is associated with the stabilized TBP-TFIIA complexes bound to the DNA. In this regard, it has previously been shown that T antigen interacts with TBP; in the present study, we show that T antigen also interacts with TFIIA in vitro. In testing the ability of T antigen to stabilize the TBP-TFIIA complex, we found that stabilization is highly sensitive to the specific sequence context of the TATA element. Previous studies showed that T antigen could activate simple promoters containing the TATA elements from the hsp70 and c-fos gene promoters but failed to significantly activate similar promoters containing the TATA elements from the promoters of the SV40 early and adenovirus E2a genes. We find that the ability to stabilize the TBP-TFIIA complex on the hsp70 and c-fos TATA elements, and not on the SV40 early and E2A TATA elements, correlates with the ability or inability to activate promoters containing these TATA elements.

FIG. 1

Simple promoters used to study the effect of SV40 T antigen on the formation of the TA and TAB complexes. The promoters shown in the top diagram were based on the promoter containing one Sp1 binding site upstream of the TATA element in the hsp70 promoter (−77 to +7 bp). Similar promoters with alternative TATA elements were constructed such that the SV40 early and adenovirus (Ad) E2a TATA elements (11 bp each) were substituted for the hsp70 TATA element (11 bp). The lower diagram shows the 6×Sp1-fos promoter, which has six Sp1 elements upstream of the fos TATA element. Both the Sp1-hsp70 TATA promoter and the 6×Sp1-fos TATA promoters are transcriptionally activated by T antigen; however, the Sp1-SV40 early TATA and Sp1-E2a TATA promoters are not transcriptionally activated by T antigen (reference and unpublished observations). Nucs., nucleotides.

FIG. 2

T antigen stimulates the formation of the TA complex on the Sp1-hsp70 TATA promoter. Various combinations of purified TBP, TFIIA, TFIIB, and T antigen (Tag) (as indicated; also described in Materials and Methods) were incubated with a ³²P-labeled Sp1-hsp70 TATA promoter fragment. The complexes formed were analyzed by EMSA.

FIG. 3

DNase footprinting analysis of the Sp1-hsp70 TATA promoter. The T-antigen (Tag)-mediated stimulation of the formation of the TA complex, shown in Fig. 2, was verified in similar binding studies analyzed by DNase footprinting. Increasing concentrations of TBP (0.5, 5, and 50 ng) were used in reactions with TFIIA, TFIIB, and T antigen (as indicated; also described in Materials and Methods).

FIG. 4

T antigen (Tag) can stabilize the TA complex on the 6×Sp1-fos TATA promoter but not on the Sp1-SV40 early TATA and Sp1-E2a TATA promoters. EMSAs identical to those described for Fig. 2 were performed for the 6×Sp1-fos TATA (A), Sp1-SV40 early TATA (B), and Sp1-E2a TATA (C) promoters.

FIG. 5

Correlation of transcriptional activation by T antigen and the stimulation of the formation of the TA complex on the four TATA elements. (A) T-antigen (T Ag)-mediated transcriptional activation of the promoters shown in Fig. 1 containing the hsp70, fos, SV40 early, and Ad E2A TATA elements. (B) T-antigen-mediated stimulation of the formation of the TA complex on the TATA elements of the four promoters shown in Fig. 1.

FIG. 6

Stabilization of HeLa D*-A complex by T antigen is TATA element dependent. The HeLa-TFIID* fraction (described in Materials and Methods and in Results) was tested for binding in the presence and absence of purified T antigen (Tag), TFIIA, and TFIIB (as indicated; see also Materials and Methods). Two different concentrations of T antigen (+ = 100 ng; +* = 200 ng) were used. Complex formation was analyzed by EMSA using the Sp1-hsp70 TATA promoter (A) and the Sp1-SV40 early TATA promoter (B).

FIG. 7

T antigen is present in the TAB preinitiation complex. A DNA immunoprecipitation assay (see Materials and Methods) were performed to determine whether T antigen (Tag) was associated with the preinitiation complexes formed on the Sp1-hsp70 TATA promoter. Binding reaction mixtures, using a ³²P-labeled promoter fragment, were prepared with the various components (as indicated). After incubation, the reaction mixtures were immunoprecipitated with an anti-T-antigen antibody to precipitate the T-antigen-containing protein-DNA complexes. The precipitated ³²P-DNA was visualized by PAGE.

FIG. 8

In vitro interaction between T antigen and TFIIA. (A) Binding reaction mixtures were prepared with the components indicated, including in vitro-synthesized, ³⁵S-labeled TFIIA (αβ and γ subunits). After incubation, the samples were immunoprecipitated with anti-T-antigen serum to precipitate the T-antigen (Tag)-containing complexes. The precipitates were analyzed by SDS-PAGE. Sizes are indicated in kilodaltons. (B) Binding of in vitro-synthesized, ³⁵S-labeled TFIIA (αβ and γ subunits) was tested in in vitro binding reactions with full-length T antigen (T Ag) fused to the glutathione binding moiety of GST or GST fusions with regions of T antigen (T1 to T5) as described in Table 2. Input lanes represent 20% of the total amount of protein put into the binding reactions. Sizes are indicated in kilodaltons.

FIG. 9

T antigen increases formation and prevents the dissociation of the TA complexes formed on the hsp70 TATA element. (A) Determination of the association rate of the TA complex in the presence and absence of T antigen. An EMSA was performed to determine the rate of association of the TA complex on the ³²P-labeled Sp1-hsp70 TATA promoter in the presence or absence of T antigen. Binding reaction mixtures were prepared, and at different time points after mixing, samples were loaded on a running gel. The intensity of the bands representing the TA complex was quantitated with a Molecular Dynamics PhosphorImager and plotted as amount of TA complex formed over time. (B) Determination of the dissociation rate of the TA complex in the presence and absence of T antigen. Binding reaction mixtures similar to those described for panel A were incubated for 1 h to bring the binding to equilibrium. Then an excess of unlabeled Sp1-hsp70 TATA promoter DNA was added as a competitor. At various time points after addition of the competitor, samples were removed and loaded on a running gel. The intesity of the bands representing the TA complex was quantitated with a PhosphorImager and plotted as a percentage of the intensity of the band at equilibrium.