Functional interaction of the bovine papillomavirus E2 transactivation domain with TFIIB

Abstract

Induction of gene expression by the papillomavirus E2 protein requires its approximately 220-amino-acid amino-terminal transactivation domain (TAD) to interact with cellular factors that lead to formation of an activated RNA polymerase complex. These interaction partners have yet to be identified and characterized. The E2 protein localizes the transcription complex to the target promoter through its carboxy-terminal sequence-specific DNA binding domain. This domain has been reported to bind the basal transcription factors TATA-binding protein and TFIIB. We present evidence establishing a direct interaction between amino acids 74 to 134 of the E2 TAD and TFIIB. Within this region, the E2 point mutant N127Y was partially defective and W99C was completely defective for TFIIB binding in vitro, and these mutants displayed reduced or no transcriptional activity, respectively, upon transfection into C33A cells. Overexpression of TFIIB specifically restored transactivation by N127Y to close to wild-type levels, while W99C remained inactive. To further demonstrate the functional interaction of TFIIB with the wild-type E2 TAD, this region was fused to a bacterial DNA binding domain (LexA:E2:1-216). Upon transfection with increasing amounts of LexA:E2:1-216, there was reduction of its transcriptional activity, a phenomenon thought to result from titration of limiting factors, or squelching. Squelching of LexA:E2:1-216, or the wild-type E2 activator, was partially relieved by overexpression of TFIIB. We conclude that a specific region of the E2 TAD functionally interacts with TFIIB.

FIG. 1

E2 interacts with immobilized TFIIB in vitro. (A) GST:TFIIB immobilized on glutathione beads was incubated with in vitro-translated ³⁵S-labeled proteins. Bound proteins were visualized and quantitated by a PhosphorImager. GST:TFIIB retained ∼10% of full-length E2 (lane 2), 6% of E2:1-216 (lane 3), and 3% of E2:162-410 (lane 4). No binding was observed between luciferase and GST:TFIIB (lane 1). (B) Binding of six-histidine-tagged E2:1-216 to GST:TFIIB deletion mutants. Wild-type GST:TFIIB and deletion mutants were incubated with 0.5 ml of crude cell extract of six-histidine-tagged E2:1-216 expressed in E. coli. Bound proteins were analyzed by SDS-PAGE and visualized by Western blotting with anti-E2 monoclonal antibody B201. E2:1-216 bound to wild-type GST:TFIIB (lane 2), Δ202-269 (lane 7), and Δ238-316 (lane 8) but not to GST alone (lane 1), the negative control GST:SD21-HC8 subunit gene of human proteasome (lane 9), and four other GST:TFIIB deletion mutants: Δ4-85, Δ45-123, Δ118-174, and Δ178-210 (lanes 3 to 6, respectively).

FIG. 2

The N-terminal activation domain of E2 interacts with TFIIB. (A) Coomassie blue-stained polyacrylamide gel of representative GST:E2 fusion proteins used in the experiments: GST (lane 1), GST:E2:74-134 (lane 2), GST:E2:54-134 (lane 3), GST:E2:1-91 (lane 4), and GST:E2:1-134 (lane 5). (B) Affinity chromatography with purified FLAG-TFIIB and GST:E2 directly demonstrates binding. Truncated forms of BPV-1 E2 were expressed and purified from E. coli (pLysS) as GST fusion proteins. FLAG-TFIIB was purified from E. coli (pLysS) with FLAG-tagged M2 beads eluted with FLAG peptides. Bound FLAG-TFIIB was detected with FLAG antibody and ECL. GST:E2:1-91 (lane 3), GST:E2:54-134 (lane 7), and GST:E2:74-134 (lane 9) bound FLAG-TFIIB, but GST (lane 1), GST:E2:1-54 (lane 2), GST:E2:15-91 (lane 4), GST:E2:15-134 (lane 5), GST:E2:54-91 (lane 6), GST:E2:74-91 (lane 8), and GST:E2:91-134 (lane 10) showed no interaction with TFIIB. (C) Specific binding of TFIIB to the E2 TAD. ³⁵S-labeled TFIIB or luciferase was synthesized in reticulocyte lysates and incubated with GST (lanes 1 and 2), GST:E2:54-134 (lanes 3 and 4), GST:E2:113-286 (lanes 5 and 6), GST:E2:215-410 (lanes 7 and 8), or GST:E2:74-134 (lanes 9 and 10). GST:E2:54-134 retained 26% (lane 3) and GST:E2:74-134 retained 9% (lane 9) of input TFIIB. (D) Summary of the E2 regions tested for binding to FLAG-TFIIB.

FIG. 3

E2 TAD mutants with reduced binding to TFIIB in vitro are also defective for TFIIB interaction in vivo. Recombinant GST:E2 fusion proteins were synthesized in E. coli, purified, and immobilized on glutathione-Sepharose beads. FLAG-TFIIB was used in panels B and D, and in vitro-translated TFIIB was used in panel C. ECL Western blotting with anti-FLAG antibody was used to detect captured FLAG-TFIIB as described in Materials and Methods. (A) Coomassie brilliant blue-stained GST fusion proteins in the GST:E2:1-286 form used in panels C and D: Q66R (lane 1), S93P (lane 2), E105G (lane 3), P106S (lane 4), N127Y (lane 5), W130R (lane 6), GST (lane 7), and wild type (lane 8). (B) FLAG-TFIIB binding to E2 mutants (in GST:E2:1-134 form) and other fragments: GST (lane 1), GST:E2:74-216 (lane 2), GST:E2:1-134 (lane 3), GST:E2:1-98 (lane 4), GST:E2:1-134W92R (lane 5), GST:E2:1-134F87S (lane 6), GST:E2:1-134Q15H (lane 7), GST:E2:1-134W99C (lane 8), GST:E2:215-286 (lane 9), GST:E2:215-410 (lane 10), and 10% input FLAG-TFIIB (lane 11). (C) In vitro-translated TFIIB binding to GST (lane 1), the negative control GST:SD21-HC8 subunit gene of human proteasome (lane 2), and the following point mutants in the GST:E2:1-286 form: Q66R (lane 3), S93P (lane 4), E105G (lane 5), P106S (lane 6), N127Y (lane 7), W130R (lane 8), and wild type (lane 9). (D) FLAG-TFIIB binding to GST:E2 mutants (GST [lane 1], GST:SD21 [lane 2], GST:E2:1-286 wild type [lane 3], and GST:E2:1-134 wild type [lane 4]) and GST:E2:1-286 form mutants (Q66R [lane 5], S93P [lane 6], E105G [lane 7], P106S [lane 8], N127Y [lane 9], W130R [lane 10], and 10% input FLAG-TFIIB [lane 11]). (E) TFIIB interacts with E2 in vivo. C33A cells were transfected with the luciferase reporter pJSLuc (three E2 binding sites) and increasing amounts of pCG:E2 wild type (wt), pCG:E2W99C, pCG:E2N127Y, and pCG:E2S181F, with or without 2 μg of pCG:TFIIB. The amount of DNA in each transfection was adjusted with the empty expression vector pCG to a total of 5 μg. The luciferase activities of reporter in the absence of E2 were normalized to 1.

FIG. 4

TFIIB functionally interacts with the E2 TAD in vivo. (A) Transactivation by LexA E2 TAD fusions. C33A cells were transfected with pcDNA3:LexA:E2:1-133 (bar 3), E2:1-216 (bar 4), or E2:162-410 (bar 5) or transfected with control plasmid pcDNA3:LexA (bar 2) without E2 with 1 μg of reporter plasmid. Luciferase activity was normalized to pcDNA3 (bar 1). (B) Overexpression of TFIIB relieves squelching by the E2 TAD. Increasing amounts of pcDNA3:LexA:E2:1-216 (0.1, 1, and 2 μg) were transfected into C33A cells with 1 μg of the pDBL8 reporter plasmid, with or without 2 μg of pcDNA3:TFIIB. Vector DNA was added as necessary to standardize the total amount of expression plasmid in each reaction.