Cell, Vol. 67, 1241-l

250, December

20, 1991, Copyright

0 1991 by Cell Press

Interaction of TFIID in the Minor Groove of the TATA Element Dong Kun and Robert Laboratory Rockefeller New York,

Lee, Masami Horikoshi, G. Roeder of Biochemistry and Molecular University New York 10021

Biology

TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyi radical footprinting assays, and by binding studies with thymine analog substituted oligonuckotldes. These results provide an explanation for TFllD-dependent DNA bending at the TATA element. TFIID binding shows phosphate contacts with the same residues that were found to be essential for TFIID interactions by methyiation and thymine-speclfk modification interference assays. Dased on previous studies implicating residues conserved between the direct repeats In DNA binding, as well as models of prokaryotic DNA binding proteins, these results also suggest a model in which the direct repeats of TFIID form two basic sntlparallel f3 ribbon arms that could contact DNA through the minor groove. Introduction At least five general transcription factors, TFIIA, -B, -D, -E, and -F, are required for accurate and efficient initiation by RNA polymerase II (for a review, see Sawadogo and Sentenac, 1990). TFIID plays a key role in initiation, since it binds to the TATA element to form a complex that nucleates the assembly of the other components into a preinitiation complex and that may be stable through multiple rounds of transcription (Nakajima et al., 1988; Van Dyke et al., 1988,1989; Buratowski et al., 1989). TFIID isthought to interact at least with TFIIA, which may stabilize its binding, and with TFIIB, which is the next factor to enter the complex(Buratowski et al., 1989; Maldonado et al., 1990). Interactions with negative cofactors that bind competitively with TFIIA have also been reported (Meisterernst et al., 1991; Meisterernst and Roeder, 1991). Components of the general transcription machinery have been shown to be highly conserved among eukaryotes. Fractions from S. cerevisiae can substitute for at least two of the HeLa factors, TFIIA and TFIID, in vitro (Buratowski et al., 1988; Cavallini et al., 1988; Horikoshi et al., 1989a; Hahn et al., 1989a). The TATA binding component of TFIID (designated as TFIIDr) has been cloned from several organisms(Cavallini et al., 1989; Eisenmann et al., 1989; Hahn et al., 1989b; Horikoshi et al., 1989b; Schmidt et al., 1989a; Gasch et al.,1990;Hoeyetal.,199O;Hoffmannetal.,199Oa,1990b; Kaoet al., 1990; Which etai., 1990; Peterson et al., 1990; Tamura et al., 1991). The 180 residue C-terminal region of TFIIDr is highly conserved (80%-100%) between species,

while the N-terminal regions diverge considerably. Structural motifs within the C-terminal domain (direct repeats, basic repeat, myc homology, and sigma homology) are even more highly conserved (Hoffmann et al., 1990b). Biochemical and genetic studies have shown that the conserved C-terminal domain of TFiiDr contains all the essential regions for DNA binding, transcription initiation, and species specificity (Horikoshi et al., 1990; Cormack et al., 1991; Gill and Tjian, 1991; Poon et al., 1991; Reddy and Hahn, 1991; Zhou et al., 1991; T. Yamamotoet al., unpublished data). The unusual binding properties of TFIIDT, requiring thermal energy and characterized by slow on and off rates (Horikoshi et al., 1988; Schmidt et al., 198913; Lieberman et al., 1991) suggest unique structural motifs in the DNA binding domain. Recent biochemical analyses indicate that TFliDr binds the adenovirus major late (AdML) promoter as a monomer (Horikoshi et al., 1990). TFiiDrdependent bending was demonstrated by a permuted binding site gel retardation assay (M. Horikoshi et al., unpublished data). Studies of nucleosomes and catabolite activator protein (CAP)-promoter complexes showed that AT-rich sequences favor minor groove compression, while GC-rich sequences favor major groove compression at points of DNA-protein contact (for a review, see Travers and Klug, 1987; Gartenberg and Crothers, 1988). In view of the observation that TFllDr induces bending at the AdML TATA element, and the presence of GC-rich clusters flanking the TATA element, it was proposed (M. Horikoshi et al., unpublished data) that TFIIDT may contact the TATA element primarily through the minor groove, and that the resulting bending enhances secondary (stabilizing) contacts in the flanking regions. Since dimethyl sulfoxide (DMS) methylates the N3 moiety of adenine and the N7 moiety of guanine in the minor and major grooves of DNA, respectively (Siebenlist and Gilbert, 1980), we used methyiation interference to determine how TFIIDr contacts DNA. TFIIDr interactions with pyrimidine residues were analyzed by a thymine-specific modification interference assay and by missing contact probing (Bruneile and Schleif, 1987; Truss et al., 1990). Additionally, ethyiation interference and hydroxyl radical footprinting assays were used to analyze TFIID7 interactions with the DNA backbone. in this paper, we demonstrate by several methods that TFIIDr interacts with the minor groove of DNA at the TATA element. In addition, we propose a model for TFIIDr binding to DNA, based on these rgsults and previous studies of other (prokaryotic) proteins that bend DNA through minor groove interactions. Results TFIIl&Purtne Contacts Methylation interference assays were performed to test the hypothesis that TFIIDr binds to the minor groove of DNA at the TATA element and thus induces DNA bending. An end-labeled DNA fragment containing the AdML pro-

Cdl 1242

a competitor

-

TFIID

-+

-

mt

wt

+

+

3

4

Figure 1. Methylation Interference of TFIIDT Binding (a) Separation of TFIIDr-DNA complexes from free DNA. Binding specificity was confirmed by coincubation of the labeled 180 bp AdML fragmentwithanunlabeledcompetitoroligonucleotide containing AdML promoter wild-type (wt, TATAAAA) or mutant-type (mt, TAGAGAA) sequences. (b and c) The 180 bp Xbal-BamHI fragment of AdML promoter, 32P-labeled on the trapscribed strand (b) or on the nontranscribed strand (c), was modified with DMS and incubated with TFIIDT. The bound and free probes, together with the input DNA, were cleaved with NaOH and analyzed on a sequencing gel. (d) Summary of methylation interference. The magnitude of each effect is shown by the size of the bar above and below that position of the DNA sequence.

TFIID-DNA+

DNA

free

+

1 2

C

c C C G A T A T T

/ :.

-35

-30 /

T T C C C C c 12

-25

-20

-25

L. -30

G G -35

-20

3

12

3

non-transcribed

transcribed

I ox

5x

n

I

;GGGGGCTAT -35

-30

C C C C C C G A

u

- 5x

T G G G G G A A A A T A T C

T

A

h AA A A -25 T T T T

II

Ill

G G G G G T G

non-transcribed

-20

C C C C C A c

transcrlbeo

o

increased

intensity

I

decreased

intensity

mater was partially modified by DMS and incubated with purified recombinant yeast TFIIDT. Bound and free probes (Figure 1a) were extracted and treated to cleave the phosphodiester backbone at the position of each methylated base. The positions of the DNA sequence at which methylation interferes with formation of the TFIIDt-DNA complex

were revealed by comparison of the cleavage patterns of DNA from the complex and from the unbound probe. Figures 1 b and 1 c present the experimental results of the methylation interference assay, and a quantitation of the results based on densitometric scanning is shown in Figure 1 d. Intensities of bands in the bound probe lanes were

TFIID 1243

Binding

to the Minor Groove

b

a

T G -20 G G G G

*II

L

C C G A

f

T -30 A

Figure 2. KMnO, Modification and Depyrimidation Interference of TFIIDr Binding (a and b) The 140 bp Xbal-Seal fragment of AdML promoter, “P-labeled on the transcribed strand (a) or on the nontranscribed strand (b), was modified by either KMnO, or hydrazine. Sound and free probes, together with the input DNA, were cleaved with piperidine and analyzed on a sequencing gel. (c) Summary of the data from KMnO, interference. The magnitude of each effect is shown by the size of the bar above and below that position of the DNA sequence.

T

T T

\T

\

1

2

3

4

-25 C C C C c -20 A 1

5

2

3

4

5

non-transcribed

transcribed

C - 5x

G G G G G G C -35

CCCCCCGATAT

T -30A T A

A A A G G G G G -25

-20

TG

T C C C C C A C

non-transcribed transcribed

u

“7

10x

o -

increasd decreased

compared with those in the free probe lane. Methylation of every conserved adenine residue in both strands of the TATA element (at positions -31 and -29 in the transcribed strand and -30, -28, -27, and -28 in the nontranscribed strand) was shown to interfere with TFllDr binding. One guanine residue at position -23 in the nontranscribed strand showed strong interference, while the other three guanine residues at positions -22, -21, and -20 in the same strand showed weak interference. However, methylation of guanine residues at the 5’ flanking GC cluster showed little effect on TFllDt binding. The guanine residues at the boundaries of the TATA element, at positions -24 in the nontranscribed strand and -32 in the transcribed strand, showed enhanced intensities in the bound probe lanes and correspondingly decreased intensities in the free probe lanes. The results presented in Figure 1 indicate that TFllDr contacts DNA through the minor groove at the TATA element and through the major groove at the downstream GC cluster flanking the TATA element, since methylation of adenine and guanine occurs in the minor and major groove surfaces of DNA, respectively.

intensity intensity

Therefore, minor groove compression at AT-rich sequences and major groove compression at GC-rich sequences by TFIIDr may induce DNA bending in a fashion similar to that shown for CAP-DNA and nucleosome complexes (Travers and Klug, 1987; Gartenberg and Crothers, 1988; Schultz et al., 1991). TFIID-Pyrlmldlne Contacts The interaction between TFllDr and thymine residues was analyzed using thymidine-specific modification with KMn04. The proposed reaction mechanism of KMnOa modification is glycosylation of the C568 double bond of thymidine in single-stranded DNA followed by oxidation to carboxylic acid and/or ring opening (Rubin and Schmid, 1980). Bound and unbound probes were prepared and the positions of thymine residues important for TFIID7 binding were analyzed as described in the methylation interference assay. Only thymine residues on the transcribed strand, at positions -28, -27, and -28, interfered with TFllDr binding following KMnO, modification (Figure 2). Consistent with this, the adenine residues complementary

Cdl 1244

transcribed strand DNA sequences

4 L 5 Gs r--h~~r-J--l mt wt mt

comoetitor

TFI ID-DNA complexes

r 5 z k

E 2 k k wt

mt

wt

E E 3 z mt

wt

4.

+

.. free

DNA

+ I

2345670

Figure 3. Effect of Substitution of Thymine Analogs on TFllDr Binding Individual thymine residues at positions -30, -25 or -27 in the transcribed strand were replaced with deoxyuridine. The DNA sequences of the TATA element in the transcribed strand of the oligonucleotides used are indicated above each lane. Radiolabeled and annealed oligonucleotides corresponding to sequences from positions -52 to +I3 of the AdML promoter and containing the substituted residue were incubated with TFIID and either wild-type (wt) or mutant-type (mt) competitor oligonucleotides. followed by electrophoresis on a nondenaturing 5% polyacrylamide gel.

to these thymines showed strong interactions with TFIIDr in the methylation interference assay (Figure Id). Results from the KMnO, interference assay were compared with those obtained from the missing contact assay. In the latter procedure, pyrimidine residues in DNA are removed by hydrazine treatment to determine the participation of individual pyrimidines in protein binding (Brunelle and Schleif, 1987). When applied to TFllDr (Figures 2a and 2b, lanes 4 and 5), this method revealed contacts on the transcribed strand at the same thymine residues detected in the KMnO, interference assay (-28, -27, and -28) as well as an additional contact at position -25. Thus, the KMn04 modification interference and missing contact assays differ slightly in the way they detect protein interactions with DNA. However, TFIIDT binding was not inhibited either by modification or by removal of thymine residues at positions -29 and -31 in the nontranscribed strand or at position -30 in the transcribed strand. Instead, the thymine residues (-25 and -30 in the transcribed strand) immediately adjacent to the GC cluster showed enhanced intensities in the bound probe lanes in the KMnO, modification interference assays (Figure 2), which indicates enhanced binding of TFIIDT. This suggests TFllDz interactions with the DNA backbone rather than the base at these positions. The enhanced binding of TFllDz by elimination of the cytosine residue at position -24 in the transcribed strand has the same explanation. Thus, in contrast to a broad spectrum of contacts at adenine residues by TFllDr (summarized in Figure Id), contacts with thymine residues are limited to a smaller region of the TATA element (summarized in Figure 2~). The missing contact assay can alsodetect interactions between protein and cytosine residues in the flanking regions of the TATA element, but almost no interference with TFllDr binding by cytosine modification was detected in either strand. Since the KMnOd modification interference assay did

not discriminate between major and minor groove contacts byTFIID7, thymine residues in the transcribed strand were replaced systematically with deoxyuridine. This removes the 5-methyl group in the major groove, which can be recognized by protein (Schleif, 1988; Luisi et al., 1991). In the case of the glucocorticoid receptor, a significant contribution of 5methyl contacts in the major groove was shown by comparison of X-ray structural analysis and binding studies with deoxyuridine substituted DNA (Truss et al., 1990; Luisi et al., 1991). Thus, substitution of deoxyuridine for a single thymine residue (designated as T12 in Truss et al. [lQQO]) that had been implicated in binding by the KMnO, modification interference assay reduced the affinity 1O-fold; this is consistent with the X-ray cocrystal structure showing van der Waals interactions between the B-methyl group of the same thymine (designated as T5 in Luisi et al. [lQQl]) and valine residue 482 in the receptor. In contrast, as shown in Figure 3 for TFIIDT interactions with the TATA element, the substitution of deoxyuridine for thymines (-28 and -27, transcribed strand) shown by the KMnOa modification interference assay to be important for TFllDr binding did not change the TFIIDT binding affinity; this indicates that the TFllDz interactions with thymine residues do not involve interactions with the 5-methyl group in the major groove. This suggests possible TFIID? interactions with either the 02 or the 04 moiety of these essential thymine residues. Given that methylation interference and hydroxyl radical footprinting (below) assays indicate TFIIDT interactions in the minor groove, an interaction with the 02 moiety (in the minor groove) is more likely. In further support of the lack of TFllDr interactions with thymine in the major groove, the substitution of deoxyuridine for thymine at position -30 in the transcribed strand showed a Cfold increase in TFllDz binding affinity (Figure 3, lane 7). This is consistent with the observation of enhanced TFIID binding following destruction of this residue by KMn04 modification (Figure 2a), and the suggestion that TFllDr recognizes the DNA backbone, rather than a base, at this particular position. TFIIDr-Phosphate Contacts To evaluate the role of phosphate groups in TFIIDT binding, DNA was ethylated with ethyl nitrosourea (Siebenlist and Gilbert, 1980). Bound and unbound probes were prepared and analyzed as described above. Figure 4 shows typical results of ethylation interference assays of TFIIDT binding. Several conclusions can be drawn from these results. There are at least five phosphate groups in the transcribed strand that interact closely with TFIID2, at positions -30, -28, -27, -28, and -25 (Figure 4a). The ethylated fragments were not resolved as distinctly as those generated by DMS or KMn04 modification, since heat and alkali can break ethylated phosphates on either side of the DNA backbone. In addition, ethyl nitrosourea modifies all four bases at a variety of positions (Sun and Singer, 1975; Sakonju and Brown, 1982). Identification of TFIIDT interactions with the phosphate residues in the nontranscribed strand was more difficult because of poor resolution. However, it is clear (Figure 4b) that TFIIDr contacts the phos-

TFllD 1245

Binding

to the Minor Groove

b

a

Figure 4. Ethylation Binding

D

r 72:s

cl .f

T A

/

z

2

-30

T G G G /”

-20

G (4 -25

Interference

of

TFIIDr

(a and b) The 140 bp Xbal-Scal fragment of the AdML promoter, gP-labeled on the transcribed (a) or nontranscribed (b) strand, was modified with ethyl nitrosourea. Samples were cleaved with NaOH and analyzed on a sequencing gel. (c) Summary of the data. The positions interfered with by ethyfation are indicated by arrows.

A

T T

T C G G G 1

2

3

4

1

transcribed

G ir G G G G C -35

2

3

-35

4

non-transcribed

T A T AA A A -30

-25

G G G G G T

non-transcribed

-20

C C C C C C G A T A T T T T C C C C C A transcribed 1‘ t-r?-t

phate group of the guanine residue (at position -23 in the transcribed strand) identified to be important for TFIIDT binding in the methylation interference assay (Figure lc). Since the bands corresponding to adenine residues within the TATA element (nontranscribed strand) in the bound probe lane show decreased intensities compared to the bands in the free probe lane (Figure 4b), it is likely that there are phosphate contacts by TFllDz at these positions. The phosphate groups that were found to interfere with TFllDr binding when ethylated are denoted by arrows in Figures 4c and 6. From the results shown here, it is possible to deduce that TFllDr shows phosphate contacts with the same or neighboring residues that were identified as essential for TFllDr binding by methylation and KMnO., modification interference assays. TFIIDr-Deoxyribose interactions TFIID7 interactions with deoxyribose were detected by hydroxyl radical footprinting. Hydroxyl radicals, generated by Fe(ll).EDTA-promoted reduction of hydrogen peroxide, are thought to attack deoxyribose, followed by breakage of the DNA backbone (Tullius and Dombroski, 1966). Since the hydroxyl radical is very small, tight contacts with protein can be detected. Hydroxyl radical cleavage is known to be very sensitive to protection or distortion of DNA in the minor groove (Van Dyke et al., 1962; Churchill et al., 1990; Oakley and Dervan, 1996). Since the concentration of hydroxyl radical cleavage reagent used by Tullius and Dombroski (1966) caused severe dissociation of TFIIDs-DNA complexes, the concentration was decreased by half to retain those complexes (see Experimental Procedures for details). By this assay, the regions with

which TFIIDT strongly interacts are restricted to the TATA element (Figure 5). Most importantly, the regions protected from hydroxyl radical cleavage on complementary DNA strands show two nucleotide offsets in the 3’direction and peaks (loci) of maximal protection that are shifted toward the 3’ boundaries; this pattern is characteristic of DNA-protein interactions in the minor groove (Van Dyke et al., 1962; Churchill et al., 1999; Oakley and Dervan, 1990). The protected regions are similar to those protected from cleavage by methidiumpropyl-EDTA.Fe(ll) with partially purified human TFllDr (Sawadogo and Roeder, 1965). Such a -10 bp protection pattern by hydroxyl radical footprinting (Figure 5c) suggests TFIIDT interactions on one face of the helix in the minor groove of DNA. The guanine residue (position -23 in the nontranscribed strand) shown to interact with TFIIDT by methylation interference (Figure lc) was not strongly protected from hydroxyl radical cleavage, presumably because the corresponding TFllDr interaction in the major groove does not cover the DNA backbone. Discussion Minor Groove Binding of TFIID7 at the TATA Element The hypothesis that TFIID interacts through the minor groove is supported by hydroxyl radical footprinting and methylation interference assays, and by binding studies with deoxyuridine substituted oligonucleotides. Hydroxyl radical footprinting analysis revealed 9-10 bp protected regions (on complementary strands) with two nucleotide offsets and peaks of maximal protection that were both

Cell 1246

Figure 5. Hydroxyl TFllDr

a &I

g k

5 s T -20 G G

r

G G

A -25 A A A T

-25

Radical

Footprinting

of

(a and b) The 140 bp Xbal-Scal fragment of the AdML promoter, =P-labeled on the transcribed (a) or on the nontranscribed (b) strand, was incubated with TFllDt and cleaved with hydroxyi radii cleavage reagente. Cleavage waa stopped by addition of l/l 5 vol of 80% glycerol and 80 mM KCI. Sound and free probes were separated on a nondenaturing gel and analped on a sequencing gel. (c) Summary of the data. The magnitude of protection is shown by the size of the bar above and below that position of the DNA sequence.

A -30 T C 71, G G

1 2 3 transcribed

-35

1 2 3 non-transcribed

C

GGGGCTATAAAAGGGGG -35 C C c c G

5xX

10x

-30

non-transcribed -25

-20

A T A T T T T c c c C C

1

shifted in the 3’ direction

o -

increased decreased

(Figure 5~). This protection

tern is typical of protein-DNA interactions groove, whereas protein interactions in

transcribed

in the

intensity intensity

patminor

the major groove show loci of maximal protection that are shifted toward the 5’ direction, and only weak protection on the distal strand of the minor groove (Tullius and Dombroski, 1988; Oakley and Dervan, 1990; Churchill et al., 1990; T. Tullius, personal communication). Therefore, the minor groove binding of TFIID is solidified by hydroxyl radical footprinting analysis. Methylation interference analyses of TFIIDT binding (summarized in Figures Id and 8) indicate two important features of TFIIDT-DNA interactions. First, TFIIDT interacts with the minor groove of DNA at the TATA element.

Second, it interacts with the major groove of DNA at the 3’ flanking GC cluster of the TATA element. These two important findings help explain the basis of TFIID4nduced DNA bending: namely, minor groove compression at AT-rich sequences and major groove compression at GC-rich sequences, as shown in CAP-DNA complexes and in nucleosomes (Travers and Klug, 1987; Gartenberg and Crothers, 1988). The methylation interference assay can reveal contacts only between TFllDr and purine residues in the TATA element. In order to detect interactions of TFllDz with thymine residues, interference assays were performed with DNA in which thymine residues were modified by KMnO,. This analysis (summarized in Figures2c and 8) revealedTFIID7

TFIID 1247

Binding

to the Minor

Groove

with phosphate groups, with subsequent stabilizing interactions involving hydrogen bonds to the N3 moiety of adenine or the 02 moiety of thymine residues.

Figure

6. Summary

of Interference

Data

The DNA helix is diagrammed in a planar representation. To mark the position of the DNA backbone, a continuous ramp connects each phosphate residue. The position of the plane of each base pair is indicated by a horizontal line. Bases at positions where methylation or KMnO, modification interfere with TFllDr binding are indicated in bold type. The guanine residue for TFllD interaction in the major groove is indicated by a bold lowercase letter. Positions at which ethylation interferes with TFllDr binding are marked by arrows.

contacts with only three thymine residues, all in the transcribed strand. In general, contact points of TFllDz in the TATA element are biased to the 3’portion. This may reflect either the intrinsic asymmetry of the TATA element or TFllDr interactions with the 3’flanking GC cluster, or both. Additional evidence for minor groove contacts, rather than major groove contacts involving the thymine 5-methyl group, was provided by binding studies with oligonucleotides containing modified major groove surfaces. Thus, the substitution of deoxyuridine for thymine residues (-28 and -27 in the transcribed strand) shown to be important for TFIIDT binding by the KMn04 modification interference assay did not decrease TFIIDT binding activity, as would have been expected if major groove interactions with the 5-methyl group of thymine were important. Given the importance of these thymine residues for TFllDr binding (Figure 2) and indications from other studies (above) of minor groove interactions, these results suggest that TFIIDT may interact with the 02 moiety of thymine in the minor groove. Interactions between TFllDz and the DNA backbone were analyzed with both ethylation interference and hydroxyl radical footprinting assays (summarized in Figure 6). The ethylation interference assay indicated that TFllDz contacts at least those phosphate groups at positions identified to be important for TFllDz binding by the methylation and KMn04 modification interference assays (-30, -28, -27, -26 and -25 in the transcribed strand and -28, -27, -26, -25, and -23 in the nontranscribed strand). Since the KMnO., modification of thymine residues at two positions (-25 and -30 in the transcribed strand) showed enhanced TFIIDr binding (Figure 2c), TFllDr appears to recognize the DNA backbone rather than the bases at these positions. This conclusion is supported by the results of the ethylation interference assay (summarized in Figure 6). Overall, the results from various interference assays suggest that TFIIDT interactions with the TATA element may involve electrostatic interactions and/or hydrogen bonds

Model for TFIIDr-DNA Complexes Most sequence-specific DNA binding proteins recognize and bind DNA through the major groove. Although a number of protein structural motifs involved in major groove binding have been determined (for a review, see Harrison, 1991) the motifs for minor groove binding are relatively undefined. Proposed structural motifs for minor groove interactions include an a helix found in the globular domain of histone Hl , an antiparallel 8 ribbon, and a special type of 8 turn (proline-rich) motif found in histone Hl termini and in several other proteins (for a review, see Churchill and Travers, 1991). One characteristic of minor groove binding proteins is low sequence specificity relative to that observed for major groove binding proteins. However, TFIID shows sequence specificity with an affinity of ~2 x 1OmgM (Hahn et al., 1989a). Since an AT to TA transversion at either the third or the sixth position in the TATA element shows a dramatic decrease of transcription activity (Wobbe and Struhl, 1990) clues regarding the sequence specificity of TFllDr might be obtained in binding studies with drugs such as Hoechst 33258 or distamycin A; these molecules bind to the minor groove of AT-rich DNA in a selective fashion but do not discriminate between A and T (Churchill and Travers, 1991). Other studies (T. Yamamoto et al., unpublished data) showed that mutations of amino acids conserved between the two direct repeats of TFllDz greatly impaired sitespecific DNA binding, while mutations of nonconserved amino acids in the direct repeats and consewed residues in the central basic (lysine) repeat did not interfere with TFllDz binding. These results suggested that at least some of the protein motifs directly involved in DNA binding are determined by conserved amino acids within the direct repeat regions. Results from interference and protection assays suggest an unusual binding strategy for recognition of the TATA element by TFIIDT, which appears to reflect unique motifs in the DNA binding domain. The capacity of TFIIDT to interact with AT-rich sequences through the minor groove, and to induce DNA bending, led to a search for other proteins that might use a similar binding strategy. Hydroxyl radical footprinting and methylation interference studies on the E. coli protein IHF (intergration host factor) suggested that the minor groove is the primary contact surface for the protein (Yang and Nash, 1989). Another common property of TFIIDT and IHF is that both proteins induce DNA bending. Although a three-dimensional structure is not available for IHF, X-ray crystallographic analysis of the closely related HU protein from B. stearothermophilus showed that positively charged residues are aligned by antiparallel 8 ribbon arms, and that a pair of two-stranded 8 ribbons emerge from the body of the protein in a way that would encircle the DNA double helix (Tanaka et al., 1984). Sequence relationships between IHF and HU family members extend over the entire protein sequence, suggesting that IHF may also recognize its specific DNA sequences

Cdl 1248

by using two 5 ribbon arms which are provided from two monomers, a and 5 (Friedman, 1988; Yang and Nash, 1989). Alignment of the amino acid sequence of IHF to that of TFIIDr showed that the sequenceof theC-terminal portion TFIIDT direct repeat 1 (8 identical and 5 conserved amino acids in a span of 18) matches the sequence of the presumptive 5 ribbon arm region of the IHF one subunit better than does the sequence of direct repeat 2 (5 identical and 3 conserved amino acids in a span of 18) (H. Nash and A. E. Granston, Cell, this issue). Coupled with results presented here, these sequence similarities lead to the following model for the TFIIDr-TATA element complex. Each direct repeat region of TFIIDT (or parts thereof) could provide a two-stranded antiparallel 5 ribbon arm such that the two 5 ribbon arms of one TFIID molecule correspond to those from heterodimeric IHF. Since runs of A/T make a narrow minor groove on account of their large “propeller twist” (angle of twist between A and T bases in the same base pair) (Dickerson and Drew, 1981; Fratini et al., 1982; Churchill and Travers, 1991) it is possible that each of the two direct repeats in TFllDr may contribute a 5 ribbon arm that can fit into the narrow minor groove of the TATA element. This model does not involve interactions of the basic repeat of TFllDr with DNA, consistent with the results of mutational analysis (T. Yamamoto et al., unpublished data). Ethylation interference analyses showed that TFIIDT contacts phosphate groups at the same residues that were found to be important for TFIIDr binding by the KMnO, modification interference assay. Thus, it is reasonable to suppose that basic amino acids in the direct repeats can be aligned along the phosphate backbone of DNA, and that TFIIDr can be fixed to DNA via hydrogen bonds involving peptide amino groups and the N3 and 02 moieties of adenine and thymine, respectively. Even though the model for the IHF-DNA complex drawn by Friedman (1988) favors wrapping the two 5 ribbon arms around the minor groove, it is difficult to exclude the possibility that the direct repeats might lie across the minor groove. Despite the attractiveness of the proposed model, its relevance remains to be determined by more direct highresolution (X-ray crystallography or NMR spectroscopy) analyses of the TFIIDr-DNA complex. The requirement of thermal energy and the slow on and off rates for TFIIDr binding suggest that TFIIDT may change its conformation upon binding to DNA. The core domain of yeast TFIIDT (missing the nonconserved N-terminal domain) has been shown to bind to the TATA element without thermal energy (Lieberman et al., 1991). This binding capability of the core domain at 0% suggests that thermal energy may normally be required to alter the position of the N-terminal domain so that the core domain can interact efficiently with the TATA element (Lieberman et al., 1991). Since the C-terminal part of each direct repeat contains part of a helix-loop-helix homology (Gasch et al., 1990) that has been implicated in protein-protein interactions, each direct repeat may undergo structural transitions important for binding to DNA.

Experimental Plasmlds

Procedures and DNA Fragments

Theseriesof plasmid DNAs with a IS7 bp fragment spanning the AdML promoter from -190 to +135 were prepared as described elsewhere (M. Horikoshi et al., unpublished data). For the chemical modification interference assays, linear plasmid DNAs were dephosphorylated, labeled with [Y-~P]ATP and T4 polynucleotide kinase, and digested with an appropriate restriction enzyme. End-labeled fragments were isolated on a 7% polyacrylamide gel. Deoxyuridine substituted oligonucleotides corresponding to sequences from positions -52 to +13 of AdML promoter were synthesized for binding studies.

Purlflcatlon

of Yeast

TFIID

Recombinant yeast TFIID was overproduced in bacteria using a bacteriophage T7expression system and purified through DEAE-and heparin-Sepharose columns as described elsewhere (M. Horikoshi et al., unpublished data). The purity of the protein was over 95%.

Chemical

Ycdltlcatlon

of DNA

Partial methylation of “P-labeled probes was performed with DMS as described by Siebenlist and Gilbert (1980). About 2 pmol of DNA in 100 pl of 50 mM sodium cacodylate, IO mM MgC&, 40 mM KCI was mixed with 1 pl of a 1 :l mixture of DMS and ethanol at room temperature for 2 min. Reactions were stopped by addition of 40 91 of DMS stop solution (1M Tris-HCI [pH 7.51, 1.5 M sodium acetate, 50 mM magnesium acetate, 1 mM EDTA, 1 M P-mercaptoethanol). DNA was precipitated and washed. Thymidine-specific modification of DNA was performed as described before (Truss et al., 1990). DNA in 5 pl of 30 mM Tris-HCI (pH 8.0) was denatured at 95OC for 2 min and cooled in its water. Reaction was initiated by addition of 20 pl of 0.25 mM KMnO, solution. Samples were incubated for 10 min at room temperature and stopped by mixing with 35 pl of DMS stop solution. DNA was precipitated with ethanol twice, rinsed, and dissolved in 25 pl of 10 mM Tris-HCI (pH 8.0) 1 mM EDTA, 50 mM NaCI. DNA was annealed by heating to 95°C for 2 min and slow cooling to room temperature. Depyrimidation of DNA was done as described before (Brunelle and Schleif, 1987). DNA solution in 20 pl of water was mixed with 30 nl of hydrazine and incubated for 5 min at room temperature. The reaction was stopped by mixing with 200 nl of hydraxine stop solution (Maxam and Gilbert, 1960) and 750 nl of ethanol. DNA was precipitated and rinsed. For the ethylation interference assay, DNA in 100 pl of 50 mM sodium cacodylate (pH 8.0) were mixed with an equal volume of ethanol saturated with ethyl nitrosourea (Siebenlist and Gilbert, 1980). The samples were incubated for 1 hr at 50°C, followed by addition of IO nl of 5 M ammonium acetate and 200 pl of ethanol to precipitate DNA. The DNAfragmentswereresuspended, reprecipitated, and rinsed with ethanol.

Interference

of TFllDr

Blndlng

Binding reactions of TFllDr were done as described before (Horikoshi et al., 1989a). Bound and free probes were separated on a nondenaturing gel (Horikoshi et al., 1989a) and identified by exposing the wet gel to Kodak XAR-5 film for 1 hr. The DNA fragments were electroeluted, extracted with phenol-chloroform, purified by ethanol precipitation, and rinsed. DNA was cleaved at the positions modified by DMS as described (Craig and Nash, 1984) with minor modifications. DNA in 44 ul of 20 mM NaOAc and 1 mM EDTA was mixed with 7.5 91 of 1 M NaOH and then heated for 30 min at 90°C. Reactions were stopped by addition of 2 nl of 1 M Tris-HCI (pH 7.0). DNA was precipitated and rinsed with ethanol. Cleavage of DNA at pyrimidine residues modified by either KMnO, or hydrazine was done in 100 nl of 1 M piperidine by heating for 30 min at 90°C. The samples were then lyophilized to remove piperidine. Ethylated DNA was dissolved in 15 91 of IO mM sodium phosphate buffer (pH 7.0) and 1 mM EDTA. Each sample was incubated with 25 pl of 1 M NaOH at 90°C for 30 min. Reactions were stopped and DNAwas precipitated. Cleaved fragments were dissolved

TFIID 1249

Binding

to the Minor

Groove

in formamide-dye mixture and separated acrylamide sequencing gel.

Hydroxyl

Fledlcel

on an 8 M urea-7%

poly-

Friedman, D. I. (1988). Cell 55, 545-554.

FootprIntIng

Hydroxyl radical footprints of yeast TFIIDT were performed using methodology described by Tullius and Dombroski (1986) with slight modifications. Briefly, hydroxyl radical cleavage reagents were allowed to react for 1 min at room temperature with DNA-protein complexes prior to electrophoretic separation. Reaction conditions for DNA-protein binding were the same as described in Horikoshi et al. (1989a), but did not contain glycerol. Final concentrations of Fe(ll), EDTA, H202, and ascorbic acid in the solutions were 50 PM, 100 uM, 0.00150/b, and 0.5 mM, respectively. The reactions were stopped by the addition of 1115 vol of 80% glycerol and 60 mM KCI and then loaded onto a native gel. The free and bound DNA were recovered from the gel as described before. All samples were extracted with phenol-chloroform, precipitated, and rinsed.

Acknowledgments We thank Jeff DeJong, Tae Kook Kim, and Jong-Bok Yoon for discussions; Alexander Hoffmann, Ernest Martinez, Camilo Parada, and Stephen Burley for critical comments on the manuscript; and Thomas D. Tullius for information on hydroxyl radical footprinting. M. H. is an Alexandrine and Alexander L. Sinsheimer Scholar. This work is supported by National Institutesof Health grants to R. G. R. and M. H. and by funds from the Pew Charitable 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 be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

Reversible bending CGCGAAITBrCGCG.

18, 1991; revised

November

19, 1991

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In Proof

The data referred to as ‘M. Horikoshi et al., unpublished now be updated: Horikoshi, M., Bertuccioli, C., Takada, J., Yamamoto, T., and Roeder, R. G. (1992). Transcription induces DNA bending upon binding to the TATA element. Acad. Sci. USA 89, in press.

data” can R., Wang, factor IID Proc. Natl.

Interaction of TFIID in the minor groove of the TATA element.

TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyl radical footprinting assays, an...
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