The BTAF1-NC2α interaction is necessary for stimulation of BTAF1 binding to TBP. BTAF1-enriched RK13 cell lysate (1 μl) and GST-TBP-coated beads were incubated in the absence (lane 3) and in the presence (lanes 4 to 9) of 100 pmol of the indicated hisNC2α mutant proteins as described in Materials and Methods. hisNC2α proteins were detected using anti-His antibodies in a Western blotting experiment.

Direct interaction of BTAF1 with NC2α but not with NC2β. (A) The BTAF1 505-891 fragment interacts directly with NC2α. GST pull-down was performed as described in Materials and Methods by using bacterial lysates containing equal amounts of GST-BTAF1 (505-891) (lane 2), GST-TBP (lane 3), and GST-ΔTFIIS (lane 4). Purified hisNC2α, hisNC2β, or NC2(α/hisβ) was added as indicated. (B) NC2 proteins were expressed and purified to near homogeneity (unpublished data). GST-NC2α, GST-NC2β, NC2(GSTα/hisβ), and NC2(hisα/GSTβ) were tested for their ability to bind BTAF1 and hisTBP. The lysate of RK13 cells overexpressing BTAF1 (3 μl; top panel) or purified hisTBP (20 ng; bottom panel) were incubated with glutathione-agarose beads coated with different NC2 proteins (as indicated above the lanes) in the absence (lanes 2 to 5) or presence(lanes 6 to 9) of 20 μg of ethidium bromide (EtBr)/ml as described in Materials and Methods. This concentration of ethidium bromide was shown to severely diminish TBP-DNA and B-TFIID-DNA interaction in EMSA (data not shown). Bound proteins were visualized by immunoblotting using TBP- or BTAF1-specific antibodies. (C) Hexahistidine pull-down was performed as described for the GST pull-down shown in panel B except that NiNTA-agarose beads, purified hisNC2α (lane 2), hisNC2β (lane 3), NC2(hisα/β) (lane 4), or NC2(α/hisβ) (lane 5) was used. GST-TBP was used as the source of TBP in the bottom panel.

NC2α stimulates the interaction of BTAF1 with TBP in an ATP-dependent manner. (A) RK13 cell lysate (1 μl) containing overexpressed BTAF1 was incubated with glutathione-agarose beads coated with GST-TBP (lanes 4 to 7) or GST-ΔTFIIS (lane 8) in the absence (lanes 4 and 8) or presence of 6.5 pmol of hisNC2α (lane 5), hisNC2β (lane 6), and NC2(α/hisβ) (lane 7) as described in Materials and Methods. Lanes 4 to 8 in the bottom panel contained 10 U of hexokinase and 10 mM glucose. (B) Hexokinase activity is necessary to abolish NC2α-mediated binding of BTAF1 and TBP. RK13 cell lysate (1 μl) containing overexpressed BTAF1 was incubated with glutathione-agarose beads coated with GST-TBP (lanes 3 to 6 and 8 to 11) or GST-ΔTFIIS (lanes 7 and 12) in the absence (lanes 3, 7, 8, and 12) or presence of 6.5 pmol of hisNC2α (lanes 4 and 9), hisNC2β (lanes 5 and 10), and NC2(α/hisβ) (lanes 6 and 11). Lanes 3 to 7 contained 10 U of hexokinase and 10 mM glucose. Lanes 8 to 12 contained 10 U of hexokinase inactivated by boiling for 3 min [hexokinase(inact)] and 10 mM glucose. (C) Excess ATP analogue inhibits NC2α-stimulated BTAF1-TBP interaction. RK13 cell lysate (1 μl) containing overexpressed BTAF1 was incubated with glutathione-agarose beads coated with GST-TBP (lanes 3 to 6) or GST-ΔTFIIS (lane 7) in the absence (lanes 3 and 7) or presence of 6.5 pmol of hisNC2α (lanes 4 to 6). Lane 5 contained 4 U of hexokinase (hexo) and 5 mM glucose. Lane 6 contained 2 mM ATPγS. All reaction mixtures additionally contained 5 mM magnesium chloride.

ATP hydrolysis by BTAF1 or NC2α phosphorylation is not required for its binding to TBP in the presence of NC2α. (A) The ATPase-deficient mutant of BTAF1 binds to TBP in the presence of NC2α. Reactions were performed as described for Fig. 4A. BTAF1-containing HeLa cell lysates were diluted with the lysate of cells infected with T7 polymerase-expressing virus to obtain equal concentrations of wild-type and mutant BTAF1. (B) ATP does not increase BTAF1-TBP interaction in the presence of NC2α. Reactions were performed as described for Fig. 4A, except that the reaction mixtures shown in the top panel received 0.5 μl of BTAF1-enriched RK13 cell lysate, and those shown in the middle and bottom panels received 0.5 μl of partially purified (purif.) hisBTAF1. Reaction mixtures shown in the bottom panel also contained 1 mM ATP, 20 U of creatine phos-phokinase, and 10 mM creatine phosphate. (C) NC2α phosphorylation is not required for BTAF1-TBP interaction. The reaction was performed as described for panel A, except that 10 times more GST-TBP, hisNC2α, and BTAF1-containing lysate was added. Twenty microcuries of [³²P]γATP and 5 mM MgCl₂ were included in all the reactions. Lane 6 included 50 μM LY194002 (LY), lane 7 contained 10 μM wortmannin (wort), lane 8 contained 50 μM PP2, and lane 9 contained 50 μM H89. The top panel represents the immunoblotting experiment, whereas the bottom panel represents the autoradiogram.

Analysis of the interaction between NC2 proteins and DNA-bound B-TFIID or TBP. (A) NC2α binds to both B-TFIID and TBP on DNA. EMSA was performed as described in Materials and Methods with 3 to 5 ng of B-TFIID (lanes 1 to 7) or 5 ng of TBP (lanes 8 to 14). Lanes 2 to 7 and 9 to 14 received increasing amounts of hisNC2α (0.135, 0.45, 1.35, 4.5, 13.5, and 45 pmol). Lane 15 received 45 pmol of hisNC2α. (B) NC2β can dissociate B-TFIID-DNA and stimulate formation of a TBP-NC2β-DNA complex. EMSA was performed as described for panel A. Lanes 2 to 7 and 9 to 14 received increasing amounts of hisNC2β (0.153, 0.51, 1.53, 5.1, 15.3, and 51 pmol). Lane 15 received 51 pmol of hisNC2β. (C) NC2 can dissociate B-TFIID-DNA complex. EMSA was performed with 3 to 5 ng of B-TFIID (lanes 1 to 9) or with 5 ng of TBP (lanes 10 to 18). Lanes 2 to 9 and 11 to 18 received increasing amounts of NC2(hisα/β) (0.72, 2.4, 7.2, 24, and 72 fmol, and 0.24, 0.72, and 2.4 pmol). (D) Analysis of the B-TFIID-NC2α-DNA complex by specific antibodies. EMSA was performed as described for panel A with B-TFIID (lanes 1 to 3) or TBP (lanes 4 to 6). Lanes 2, 3, 5, and 6 received 4.5 pmol of hisNC2α. Lanes 3 and 6 received 500 ng of anti-NC2α antibody. (E) BTAF1 is absent from the TBP-NC2-DNA complex. EMSA was performed as described for panel D. Lanes 2, 3, 5, and 6 received 0.24 pmol of NC2(hisα/β). Lanes 3 and 6 received 1 μg of anti-BTAF1 antibody PF299. Abbreviations: T, TBP; BT, B-TFIID; α, NC2α; β, NC2β; αβ, NC2 complex; Abα, anti-NC2α antibody; AbB, anti-BTAF1 antibody; NS, nonspecific interaction.

NC2α interacts with BTAF1 in the yeast two-hybrid system. (A) S. cerevisiae strain EGY48 was transformed with plasmids expressing LexA fusions of the human BTAF1 fragments indicated, a B42 fusion with full-length NC2α, and pSH18-34 LacZ reporter plasmid. To score for the interacting proteins, the indicated yeast strains were grown on plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) as a β-galactosidase substrate. (B) Yeast strain EGY48 was transformed with a plasmid expressing a LexA fusion of human BTAF1 (505-1133), pSH18-34 plasmid, and B42 fusions of full-length NC2α, NC2β, or the indicated NC2α mutants. β-Galactosidase activity in the cell lysates was expressed as the induction relative to the activity in lysates of yeast containing the empty B42 vector control, pSH18-34, and the LexA-BTAF1 (505-1133) plasmid. The standard deviation was less than 30% in each case. To the right are schematic representations of the NC2α fragments for each B42 construct. The different domains are indicated as follows: ac, acidic domain; P-rich, proline-rich domain. (C) The carboxyl-terminal acidic domain of NC2α is highly conserved in most metazoans. Alignment of NC2α from various organisms was performed using the Clustal W program (41). The invariant EDYD motif is highlighted. H. sapiens, Homo sapiens; M. musc., Mus musculus; D. rerio, Danio rerio; X. laevis, Xenopus laevis; D. melan., D. melanogaster; Z. mays, Zea mays; O. sativa, Oryza sativa; A. thaliana, Arabidopsis thaliana. (D) Expression of B42 fusion proteins in yeast lysates. Equal amounts of yeast cells were lysed, and levels of B42-NC2 subunit expression were visualized by immunoblotting with anti-HA antibodies. To the left of the panel the molecular weights of comigrated marker proteins are indicated.