Polymerase (Pol) III TATA box-binding protein (TBP)-associated factor Brf binds to a surface on TBP also required for activated Pol II transcription

Abstract

The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription. Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions. A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G. Kassavetis, C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek, Mol. Cell. Biol. 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment. Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription. These results indicate that Brf and TFIIB interact differently with TBP. An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs. Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.

FIG. 1

EMSAs of hTBPm3 and mutant R235E plus Brf and B" with a yeast U6 snRNA TATA box region probe. Results for hTBPm3 and R235E are shown at top and bottom, respectively. In each panel, the first three lanes show DNA-protein complexes formed with 2 ng of TBP, 6 fmol of Brf, and 88 fmol of B", as indicated at the top of the autoradiogram. The six lanes on the right, analyzed on a separate gel, show binding with hTBPm3 or R235E alone (2 ng) and with 1.1, 3.3, 9.8, 29, and 88 fmol (from left to right) of recombinant Brf. The positions of DNA-protein complexes are indicated.

FIG. 2

Brf binding to representative human TBP mutants. For the top three panels of EMSAs, 2 ng of hTBPm3 or the indicated mutant protein (whose presence is indicated by T above each lane) was incubated with U6 probe alone (left lanes) or with 1.1, 3.3, 9.8, 29, or 88 fmol of recombinant Brf (in the successive five lanes). For the bottom panel of EMSAs, 2 ng of hTBPm3 or mutant TBP was incubated with U6 probe alone (left lanes) or with 3.7, 11, 33, or 100 fmol of recombinant Brf (in the successive four lanes).

FIG. 3

DNase I footprinting activity of TBP mutants on the adenovirus 2 E4 promoter. For each mutant, three titrations (2, 4, and 8 ng, from left to right) of TBP were assayed for DNase I footprinting activity. The protected TATA box is indicated at the right.

FIG. 4

Locations of human TBP residues where mutations decrease the binding of Brf. At the top, the TBP molecule is viewed from its upstream-facing surface (relative to Pol II transcription on the adenovirus 2 major late promoter), with the N-terminal repeat to the right. DNA is shown as a wire diagram. At the bottom, TBP is shown rotated 90° so that the top of the molecule is viewed with the N-terminal repeat to the right. Residues where mutations reduce Brf binding in EMSA are darkened and designated.

FIG. 5

EMSAs with a C-terminal fragment of Brf. Two nanograms of hTBPm3, Q242E, K243E, L185E, and R186E were incubated with U6 TATA box probe and 0, 6.7, 20, 60, 180, and 540 fmol of a C-terminal fragment of Brf extending from amino acid residue 317 to the C terminus, at residue 596.

FIG. 6

EMSAs with mutant yeast TBPs. The wild type or the indicated mutant yeast TBPs were incubated with U6 TATA box probe, 100 fmol of Brf, 150 fmol of B", and increasing amounts (0.125, 0.25, 0.5, 1, 2, and 4 ng) of recombinant wild-type and mutant TBPs, as indicated.

FIG. 7

U6 snRNA gene transcription in vitro. (A) Reactions with human TBPs. All reaction mixtures contained constant amounts of hTBPm3 or the indicated TBP mutant and constant amounts of B" and yeast RNA Pol III. Either 0 (first lane) or 2.4, 7.3, or 22 fmol (from left to right) of Brf were added. Transcription products were analyzed by electrophoresis in a polyacrylamide gel followed by autoradiography. The band near the middle of the gel is in vitro-synthesized U6 snRNA. (B) Reactions with human TBP hTBPm3 or triple mutant E284R + E286R + L287E. Reaction mixtures were as described for panel A except that they contained 0, 2.4, or 7.3 fmol of Brf. (C) Reactions with yeast TBPs. For the wild type (WT) and each mutant TBP, transcription reactions were performed as described for panel A with 2.4 and 7.3 fmol of Brf.

FIG. 8

Basal in vitro Pol II transcription with human TBPs. Basal in vitro Pol II transcription from the adenovirus 2 major late promoter with a G-less cassette template was performed as described in Materials and Methods by using nuclear extract (Nxt) (lane 1), heat-treated nuclear extract (lane 2), or hTBPm3 or the indicated TBP mutant added to the heat-treated nuclear extract (lanes 3 to 9).