©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
TATA-binding Protein Residues Implicated in a Functional Interplay between Negative Cofactor NC2 (Dr1) and General Factors TFIIA and TFIIB (*)

Tae Kook Kim (§) , Yingming Zhao , Hui Ge , Richard Bernstein , Robert G. Roeder (¶)

From the (1) Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The TATA-binding protein (TBP) plays a key role in transcription initiation. Several negative cofactors (NC1, NC2, and Dr1) are known to interact with TBP in a manner that prevents productive interactions of transcription factors TFIIA and TFIIB with promoter-bound TBP. To gain insights into the regulatory interplay on the surface of TBP, we have employed mutant forms of TBP to identify amino acid residues important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB. The results show the involvement of distinct domains of TBP in these interactions. Residues (Lys-133, Lys-145, and Lys-151) in the basic repeat region are important for interactions with NC2, as well as with TFIIA (Buratowski, S., and Zhou, H. (1992) Science 255, 1130-1132; Lee, D. K., DeJong, J., Hashimoto, S., Horikoshi, M., and Roeder, R. G. (1992) Mol. Cell. Biol. 12, 5189-5196), whereas a residue (Leu-189) in the second stirrup-like loop spanning S2` and S3` is required for interaction with TFIIB. In addition, we demonstrate that NC2 is identical to the previously cloned negative cofactor Dr1. The implications of these results for TBP structure and function are discussed.


INTRODUCTION

Transcription initiation on genes encoding eukaryotic mRNA involves assembly of a functional preinitiation complex containing general initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ) and RNA polymerase II (reviewed in Refs. 3 and 4). The first step is the binding of the TATA-binding subunit (TBP)() of TFIID to the TATA box of the promoter. This step may be facilitated by TFIIA (5) and results in a stable complex, which subsequently promotes the stepwise assembly of TFIIB, TFIIF/Pol II, TFIIE, TFIIH, and TFIIJ into the promoter complexes. Consistent with the central role of TFIID (via TBP) in recruiting other general factors to the promoter complex, both TBP and TBP-associated factors within TFIID have been implicated as a target for regulatory factors (reviewed in Ref. 6). As examples most relevant to the present study, interactions of TBP (or TFIID) with promoter DNA (7, 8, 9, 10, 11) , with TFIIA (12, 13) , and subsequently with TFIIB (14, 15, 16, 17) have been shown to be regulated by various activators. Moreover, several negative cofactors ( e.g. NC1, NC2, Dr1, and ADI) may be involved in these regulatory interactions as well. By competing with TFIIA and/or TFIIB for binding to TBP, they can prevent formation of a functional preinitiation complex (18, 19, 20, 21, 22, 23) . Thus, it is possible that some activators and repressors might regulate transcription by directly modulating interactions of TBP with negative cofactors versus TFIIA and/or TFIIB. To understand these regulatory interactions within TBP, it is important to first identify the domains (or residues) of TBP that carry out each of these interactions. Here, we have used site-directed TBP mutants to define binding sites on TBP for the negative cofactor NC2 and the general factor TFIIB. These analyses, along with previous studies of TBP mutant interactions with TFIIA (1, 2) , indicate that both NC2 and TFIIA interact with the basic repeat region, whereas TFIIB interacts with a distinct domain defined, at least in part, as the second stirrup-like loop region of TBP. Furthermore, we demonstrate that NC2 is identical to the factor described as Dr1.


EXPERIMENTAL PROCEDURES

Purification of Recombinant Proteins

Dr1, Wild-type, and mutant yeast TBPs (17) , as well as yeast and human TFIIBs (24) , were expressed from the T7 polymerase vector pET11d as hexahistidine fusion proteins in bacteria and were purified by nickel chromatography (25) . GAL4-VP16 was expressed in and purified from bacteria as described (26) .

Preparation of Extracts and Transcription Factors

HeLa nuclear extracts were prepared according to Dignam et al.(27) . General transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) and the USA cofactor fraction were purified from HeLa extracts as described (18) with some modifications (28) . Sp1 was separated from the general transcription factors and purified by affinity chromatography (18) . Purification of the negative cofactor NC1 was as described (29) . The negative cofactor NC2 was purified from HeLa cell S100 extracts through phosphocellulose (P11), DEAE-cellulose (DE52), DEAE-Sephadex, S-Sepharose, Mono-S, and Mono-Q chromatographic steps as described (19) . Yeast extracts were prepared according to Kim et al.(17, 30) . Yeast basal transcription factors and mediator were prepared by chromatography of yeast whole cell extracts on Bio-Rex 70 (Bio-Rad) and DE52 (Whatman). Fraction C was prepared from yeast nuclear extracts as described (17) .

Electrophoretic Mobility Shift (EMSA) and Immunoblot Assays

The DNA fragment used for the EMSA contained the TATA box and flanking regions of the adenovirus major late promoter (positions -136 to +46). The formation of TBP-TFIIB-DNA (21) and TBP-NC2-DNA (19) complexes was determined by EMSA as described. Dephosphorylation of purified NC2 was performed as described (20) . Immunoblotting was done following the manufacturer's instructions (Amersham Corp.).

Human and Yeast in Vitro Transcription Assays

Human factor assays (25 µl) contained 1 µl of TFIIA (Mono-Q fraction, 0.5 mg/ml), 10 ng of recombinant TFIIB, 1 µl of TFIID (Mono-S fraction, 0.5 mg/ml), 1 µl of TFIIE/F/H (Mono-S fraction, 0.3 mg/ml), 0.5 µl of RNA polymerase II (Mono-Q fraction, 0.3 mg/ml), 1 µl of USA (heparin-Sepharose fraction, 0.3 mg/ml), and appropriate DNA templates (pGHMCAT, pMHIVMT, and pML53) in the presence or absence of other tested components and were analyzed as described (28) . Yeast transcription assays were performed with reconstituted fractions and DNA template (pGAL46) and analyzed as described before (17, 30, 31, 32) .


RESULTS AND DISCUSSION

The conserved core domain of TBP is known to fold into a highly compact and symmetrical structure (33) . Analyses of the TBP core domain have suggested that most of the described deletion mutants of TBP, as well as chimeras constructed with yeast and human TBP fragments, cannot adopt the proper compact three-dimensional fold of TBP (34) . Thus, in the present study, single point mutants have been employed to define amino acid residues (or domains) within TBP, which are important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB. Since lysine or leucine residues are distributed extensively in the conserved carboxyl-terminal core region, we earlier generated and analyzed a series of TBP point mutants containing either leucine to lysine or lysine to leucine changes (17, 31, 35) . With knowledge of the three-dimensional structure of TBP (33) , the present analysis has focused on TBP mutants whose residues are at exposed positions that would allow direct protein-protein interactions. Based on previous studies (17) , TBP mutants defective in both basal and activated transcription are excluded because they could simply reflect gross structural perturbations that disrupt all functions, and indeed most of these mutations map to the hydrophobic core that would be expected to disrupt the structure (33) . Selected mutant and wild-type TBPs were expressed as hexahistidine fusion proteins in bacteria, at comparable levels (data not shown), and purified by nickel column chromatography to about 80% purity (Fig. 1). Each purified TBP protein was then employed in various in vitro assays.


Figure 1: Purified TBP mutants, yeast TFIIB, human TFIIB, and human NC2. Individual purified proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue staining. Positions of protein molecular mass markers are indicated as kDa.



Basic Repeat Region of TBP Is Important for Interaction with NC2

To determine the domain of TBP important for interaction with the negative cofactor NC2, we utilized TBP point mutants and NC2 purified from HeLa cell nuclear extracts (Fig. 1) to analyze TBP-NC2 complex formation on promoter DNA by EMSA (19) . An analysis of 13 mutants showed that three TBP mutants (K133L, K145L, and K151L) were defective in TBP-NC2-DNA complex formation (Fig. 2) while maintaining full activity in TBP-DNA complex (Fig. 2) and in TBP-TFIIB-DNA complex (see Fig. 6) formation. The ability of these mutants to form normal levels of TBP-DNA complexes but not NC2-TBP-DNA complexes is more evident in the dose-response analysis of Fig. 3. Thus, three individual point mutations specifically abolished TBP interactions with NC2 on the promoter DNA. The mutated residues (Lys-133, Lys-145, and Lys-151) map to the basic repeat region in the three-dimensional structure of TBP (33) , indicating the importance of this domain for direct interactions with NC2 (Fig. 8). Other mutants with changes outside the basic repeat region showed normal TBP-NC2-DNA complex formation (Fig. 2). In support of the idea that interaction of NC2 with the basic repeat region of TBP is of functional relevance, mutants with alterations in several different basic residues (Lys-145 and Lys-151 as well as Arg-141, which is also important for NC2 interactions) were resistant to transcriptional repression by NC2 (data not shown).


Figure 2: TBP mutants defective in interactions with the negative cofactor NC2. Electrophoretic mobility shift assays contained the radiolabeled DNA probe and proteins as indicated. Specific TBP- and TBP-NC2-promoter complexes are indicated. Since slightly variable amounts of TBP were used in the reactions, the minor variations in band densities (other than those in lanes10, 13, and 14) do not reflect varying abilities of TBP mutants to form TBP-NC2-DNA complexes. In particular, to confirm defective NC2 interactions in three TBP mutants (K133L, K145L, and K151L), we employed approximately 3.5-fold higher TBP concentrations (3.5 ng) in lanes10, 13, and 14, as compared with wild-type TBP (1.0 ng).




Figure 6: TBP mutants defective in interactions with yeast TFIIB (yTFIIB). Electrophoretic mobility shift assays were performed with radiolabeled DNA probe, wild-type or mutant TBPs, and yeast TFIIB as indicated. The specific TBP-yTFIIB-DNA complexes are indicated on the side. Under these conditions, stable TBP binding requires the presence of other factors such as TFIIB (21).




Figure 3: Defective NC2 interactions in TBP mutants K133L, K145L, and K151L. Electrophoretic mobility shift assays contained the radiolabeled DNA probe and proteins as indicated. Equivalent amounts of mutant or wild-type TBP (1.5 ng) were incubated alone ( lanes2, 5, 8, 11) or in increasing concentrations of NC2 (in ng 10). The positions of the resulting protein-promoter complexes are indicated.




Figure 8: Schematic drawing of selected TBP point mutants. TBP is a highly compact and symmetric structure, resembling a saddle (33). The -carbon backbone is shown as a solidwhiteline. Also shown are the side chains of positively charged lysine residues (K133L, K138L, K145L, K151L, and K156L) in the basic repeat region and of the residue (Leu-189) located in the second stirrup-like loop spanning S2` and S3`. Three mutants (K133L, K145L, and K151L) are defective in interactions with NC2, whereas the L189K mutant is defective in TFIIB interactions. Previously, mutants K138L and K145L were shown to be defective in interactions with TFIIA (1, 2) and a yeast ATP-dependent inhibitor of TBP binding to DNA (21). In addition, mutations K156L and K138L were reported to specifically affect transcription by RNA polymerases I and III, respectively (31).



TFIIA Competes with NC2 for Binding to the Basic Repeat Region of TBP

To examine the specificity of interactions between purified NC2 and TBP, we challenged TBP-NC2-DNA complexes with TFIIA. As shown in Fig. 4 a, addition of increasing concentrations of TFIIA to a preformed TBP-NC2-DNA complex transformed the latter into a TBP-TFIIA-DNA complex ( lanes4-8). These observations are in good agreement with previous results (19, 20) and support the view that NC2 and TFIIA interact with identical or closely overlapping domains of TBP. The fact that both NC2 (above) and TFIIA (1, 2) interact with the basic repeat region of TBP is consistent with this possibility. Interestingly, the present (Fig. 2) and previous (2) analyses of TBP point mutants indicate that some basic repeat residues are specifically important for interaction with NC2 (K133L and K151L) or TFIIA (K138L), with one residue (K145L) identified as being involved in interactions with both NC2 and TFIIA. This suggests that the basic repeat region may contain distinct but overlapping sites for interactions with NC2 and TFIIA.


Figure 4: Effect of TFIIA on NC2-TBP interactions and identity of NC2 and Dr1. a, electrophoretic mobility shift assays with TBP, NC2, and TFIIA. TBP (1.0 ng) and NC2 (20 ng) were incubated alone ( lane1), in the presence of 2 units of calf intestine phosphatase ( lane2), 20 µg of bovine serum albumin ( lane3), or increasing concentrations of TFIIA (31.3, 62.5, 125, 250, and 500 ng of total protein in lanes 4, 5, 6, 7, and 8, respectively). For the competition analyses in lanes4-8, TFIIA was added after preincubation of TBP and NC2. The positions of the resulting protein-promoter complexes are indicated. b, immunoblotting analysis of NC2 and Dr1. NC1 and NC2 were prepared as described under ``Experimental Procedures.'' A fraction eluted at 0.5 M KCl from phosphocellulose and subsequently at 0.3 M KCl from DEAE-cellulose (P11/0.5-DE/0.3) was used as a control. Proteins were separated by 15% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with anti-Dr1 antisera. Positions of protein molecular mass markers are indicated as kDa. c, electrophoretic mobility shift analysis of TBP-NC2-promoter complexes with anti-Dr1 antisera. Reactions contained radiolabeled DNA probe and TBP ( lane1) or TBP plus NC2 ( lanes2-4). For the analysis in lanes3 and 4, preimmune serum or anti-Dr1 antiserum was preincubated with NC2 before testing for DNA binding with TBP. Specific TBP- and TBP-NC2-promoter complexes are indicated on the side.



NC2 Is Identical to Dr1

The initial description of NC2 (19) as a transcriptional inhibitor that blocks association of TFIIA with TBP reflects closely the situation reported later for Dr1 (20) . The latter study also demonstrated that phosphorylation of Dr1 affected its interaction with TBP on the DNA (20) . Based on these properties, the effect of NC2 dephosphorylation on its binding with TBP was determined. As shown in Fig. 4a, treatment of NC2 with calf intestine phosphatase ( lane2versuslane1) but not with bovine serum albumin ( lane3) abolished its ability to interact with TBP. Consistent with this suggestion that NC2 might have an activity similar to Dr1, immunoblotting analyses showed that highly purified NC2 but not the negative cofactor NC1 contains a protein that is recognized by anti-Dr1 antisera (Fig. 4 b, lane2). This polypeptide corresponds to the dominant band in the SDS-polyacrylamide gel electrophoresis analysis of NC2 shown in Fig. 1and its molecular weight appears almost identical to that of the major recombinant Dr1 polypeptide (Fig. 4 b, compare lane2 with lane1). Furthermore, NC2-TBP-DNA complex formation was specifically abolished by anti-Dr1 antisera (Fig. 4 c, lane4) but not by preimmune sera ( lane3). We also observed that the NC2-mediated transcriptional repression (below) can be relieved by anti-Dr1 antisera (data not shown). Altogether, these results suggest that the NC2 and Dr1 might be identical proteins, although it cannot be excluded that NC2 contains a functional polypeptide(s) in addition to that which corresponds to the recombinant Dr1. The apparently different mobilities of NC2-TBP (19) and Dr1-TBP (20) complexes, when compared with that of TFIIA-TBP complexes (Fig. 4 a), may be due to variations in the efficiency of formation of multimerized complexes of NC2 (Dr1) under different reaction conditions (19, 20) . At the same time, we cannot exclude the possibility that the NC2 and Dr1 preparations differ in some way ( e.g. in the level of phosphorylation) that alters the efficiency of multimerization, especially since phosphorylation is known to be essential for formation of the higher order complexes (20) . Related to this, even under our reaction condition (19) some NC2-TBP complexes run slower than TFIIA-TBP complexes, as reported for Dr1-TBP complexes (20) , when a much higher concentration of NC2 is used (data not shown).

NC2 Represses Transcription Both in the Yeast and the Human in Vitro Systems

We next analyzed the effects of NC2 on basal and activated transcription in both yeast and human in vitro systems. As shown in Fig. 5, the addition of increasing amounts of NC2 to the yeast in vitro reconstituted system resulted in inhibition of both basal and activator (VP16)-dependent transcription. Similar repressive effects of NC2 were observed for both basal (pML53 template) and activated transcription by GAL4(1-147)-VP16 (pGHMCAT template) and Sp1 (pMHIVMT template) in the human system reconstituted with highly purified factors (Fig. 5, b and c). The previous result (19) of a selective effect of NC2 on basal transcription may reflect the use of a less purified system in which another factor(s) may facilitate the observed discrimination. In the present study, repression was observed with various TATA box-containing promoters in both yeast and human systems. Thus, it seems likely that repression by NC2 is a general mechanism, possibly competing with general factors TFIIA and/or TFIIB to inhibit formation of functional preinitiation complexes.


Figure 5: Repression of transcription by NC2 in yeast and human in vitro systems. a, yeast in vitro reconstituted systems containing pGAL4X6 templates were incubated with variable amounts of NC2 (20 and 100 ng of total protein) in the absence ( lanes2 and 3) or presence ( lanes5 and 6) of GAL4(1-147)-VP16 as indicated. b, reactions reconstituted with human transcription factors and pGHMCAT templates were incubated with variable amounts of NC2 (10 and 50 ng of total protein) in the absence ( lanes1-3) or presence ( lanes4-6) of GAL4(1-147)-VP16 as indicated. c, reactions reconstituted with human transcription factors were incubated with pMHIVMT (containing three Sp1 sites) and pML53 (adenovirus major late core promoter) templates in the presence of Sp1. Transcription reaction conditions were the same as in b.



Distinct Regions of TBP Are Involved in Interactions with TFIIB and NC2

Apart from their ability to prevent or reverse formation of a TBP-TFIIA-promoter complex, NC2 (19) and Dr1 (20) can also displace TFIIB from TBP-TFIIB-promoter complexes. Hence, it seemed possible that TFIIB and NC2 also might interact with the same or overlapping surface(s) of TBP. To define the region of TBP important for interaction with TFIIB, we analyzed TBP-TFIIB-DNA complex formation with TBP mutants and TFIIB. Under these particular conditions, TBP alone failed to form a DNA complex, but a TBP-TFIIB-promoter complex (21, 36) was readily observed with TFIIB addition (Figs. 6 and 7). These analyses demonstrated that all but one of the tested TBP mutants, including K133L, K145L, and K151L, form TBP-yeast TFIIB-DNA complexes comparable with that displayed by wild-type TBP (Fig. 6). In contrast, mutant L189K was defective in TBP-yeast TFIIB-DNA complex formation (Fig. 6, lane16; Ref. 17). A similarly defective TBP interaction was observed with human TFIIB on the promoter DNA by electrophoretic mobility shift assays (Fig. 7). Since the L189K mutant can interact with DNA under different conditions (35) and since it can form TBP-TFIIA-DNA (2) and TBP-NC2-DNA (see Fig. 2) complexes as efficiently as does wild-type TBP, this mutation appears to specifically affect interactions with TFIIB. The failure of the L189K mutant to interact with both yeast and human TFIIB suggests that the same (or overlapping) regions of both yeast and human TBP containing a Leu-189 residue are involved in interactions with TFIIB. In the three-dimensional structure of TBP, the L189K mutation is found within the loop connecting strands S2` and S3` (Fig. 8). This loop region is at a highly exposed position that may be accessible for direct protein-protein interactions. Despite the involvement of distinct TBP regions in interactions with TFIIB and NC2, NC2 has been shown to dissociate TFIIB from the TBP-TFIIB promoter complexes. This suggests that interaction of NC2 with TBP might result in a TBP conformational change that affects TBP-TFIIB interactions. Related to the possibility of dynamic changes in TBP-promoter complexes during preinitiation complex assembly, the region containing Leu-189 has been demonstrated to be important for an activation-specific TBP-TFIIB complex, which is induced (or stabilized) by acidic activators in the yeast in vitro transcription system (17) . Recently, we have shown using a yeast in vitro system that L189K supports basal but not activated transcription, while K133L, K145L, and K151L, which are important for NC2 interactions (see Figs. 2 and 3), can support normal levels of both basal and activated transcription (17) . The fact that the L189K mutant shows normal basal transcription activities, despite its deficiency in forming TBP-TFIIB complexes, must reflect the presence of compensatory stabilizing interactions with other basal factors under transcription conditions (17) .


Figure 7: TBP mutants defective in interactions with human TFIIB (hTFIIB). Electrophoretic mobility shift assays were performed with radiolabeled DNA probe and wild-type or mutant (L189K) TBPs in the absence or presence of hTFIIB. The arrow indicates the position of the specific TBP-hTFIIB-DNA complex.



In contrast to the situation with TFIIB, NC2 and TFIIA interact with the TBP basic repeat region, which represents the convex surface of TBP-spanning -helix H2 and -strand S1` (Fig. 8). In addition, the basic repeat region has been shown to be important for interactions with several other regulatory factors (reviewed in Ref. 37), which include the adenovirus E1a activator (38) . Interestingly, E1a is capable of preventing and/or reversing the TBP-Dr1 (NC2) interaction and the corresponding repression of transcription (20, 39) . These results (see also Ref. 19), along with the present data, suggest that certain activators and/or positive cofactors could, in some situations, reverse the action of negative cofactors such as NC2 (Dr1) and thus increase promoter activity. Furthermore, the present results indicate that the basic repeat domain of TBP may be directly involved, at least in part, in the complex interplay of different sets of transcriptional regulatory factors. Recently, this region has been demonstrated to be important for transcription by RNA polymerases I and III in addition to RNA polymerase II (Ref. 31 and references therein). Clearly, the mutants described here, along with previously identified TBP mutants (reviewed in Ref. 37), will be useful for a further dissection of the functional interactions of transcription factors on the surface of TBP. As suggested by the present results and given the somewhat limited protein binding surface of TBP, many of the TBP interaction sites may be overlapping, and competition for regulatory factors could influence transcription. At the same time, TBP structural changes induced by interacting factors might modulate its ability to form preinitiation complexes. Therefore, elucidation of the interplay of various regulatory factors with TBP and their specific effect(s) on TBP-promoter complexes during preinitiation complex assembly will be critical for understanding transcriptional regulatory mechanisms.


FOOTNOTES

*
This study was supported by grants from the National Institutes of Health and the Human Frontiers Science Program (to R. G. R.) and the Pew Trusts (to The Rockefeller University). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

To whom correspondence should be addressed.

The abbreviations used are: TBP, TATA-binding protein; EMSA, electrophoretic mobility shift assay; WT, wild type.


ACKNOWLEDGEMENTS

We thank D. Reinberg for providing recombinant Dr1 and anti-Dr1 antisera; D. B. Nikolov and S. K. Burley for the figures showing the structure of TBP and S. Stevens for a critical reading of the manuscript.


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