Cell, Vol. 61. 1171-1178,

June 29, 1990, Copyright

0 1990 by Cell Press

Analysis of Structure-Function Relationships of Yeast TATA Box Binding Factor TFIID Masami Horikoshi,’ Tohru Yamamoto, Yoshiaki Ohkuma,” P. Anthony Weil,t and Robert G. Roeder’ * Laboratory of Biochemistry and Molecular Biology The Rockefeller University New York, New York 10021 t Department of Molecular Physiology and Biophysics Vanderbilt University School of Medicine Nashville, Tennessee 37232

Summary A systematic series of N-terminal, C-terminal, and internal deletion mutants of S. cerevisiae TFIID were expressed in vitro and tested for TATA box binding and basal level transcription activities using, respectively, DNA mobility shift and in vitro transcription assays. The domains responsible for these activities were colocalized to a surprisingly large region containing C-terminal residues 63-240. This region was noted previously to contain potentially interesting structural motifs (central basic core, direct repeats, and sigma factor homology) and, more recently, to be highly conserved among TFIID from different species. Deletion mutant cotranslation studies revealed that TFIID binds DNA as a monomer. The implications of these results for TFIID structure and function are discussed. Introduction The majority of promoters for genes transcribed by RNA polymerase II contains a TATA box (consensus TATAAAA) 2!j nucleotides upstream of the transcription initiation site (for review see Breathnach and Chambon, 1981). Mutagenesis experiments have shown that this element is an important determinant for both the site of initiation and the basal level of promoter activity (for review see Nakajima et al., 1988). A general transcription factor (Matsui et al., lg80) that binds to theTATA box has been identified in Drosophila (Parker and Topol, 1984), human (Davison et al., 1983; Sawadogo and Roeder, 1985; Nakajima et al., 1988), and yeast (Buratowski et al., 1988; Cavallini et al., 1988; Horikoshi et al., 1989a; Hahn et al., 1989a) cell extracts. This factor, termed TFIID (Matsui et al., 1980), is absolutely required for in vitro transcription initiation for all class II genes tested (Nakajima et al., 1988). Although other general transcription components (Matsui et al., 1980) also participate in transcription initiation complex formation (for review see Saltzman and Weinmann, 1989), TFIID plays an especially critical role in promoter activation because it binds to the TATA box independently (Nakajima et al., 1988; Sawadogo and Roeder, 1985) prior to the other general initiation factors and RNA polymerase II (Fire et al., 1984; Reinberg et al., 1987; Van Dyke et al., 1988; Horikoshi et al, 1988b; Buratowski et al., 1989). Moreover, TFIID binding to the TATA element results in

template commitment (Davison et al., 1983; Fire et al., 1984; Buratowski et al., 1988; Cavallini et al., 1988; Van Dyke et al., 1989) and may be stable through multiple rounds of transcription (Van Dyke et al., 1988, 1989). Recent biochemical analyses indicate that TFIID plays a critical role in mediating the modulation of transcription in response to regulatory factors. Thus it has been observed that: TFIID interacts cooperatively with the cellular transcriptional activators USF (Sawadogo and Roeder, 1985; Sawadogo, 1988), GAL4 (Horikoshi et al., 1988a), and ATF (Horikoshi et al., 1988b; Hai et al., 1988), leading in the latter case to more efficient binding of TFIIB, TFIIEIF, and RNA polymerase II to the promoter; a herpesvirus (pseudorabies) immediate-early protein enhances transcription in vitro by facilitating TFIID binding to the promoter (Abmayr et al., 1988; Workman et al., 1988); stable binding of TFIID to the promoter precludes chromatinmediated repression of transcription (Workman et al., 1988,199O; Workman and Roeder, 1987); and the homeodomain protein Engrailed has the potential to repress transcription by competing with TFIID for binding to TATA box (Ohkuma et al., 1990). Thus, in several systems, the interaction of TFIID with the TATA box has been implicated as a target for regulatory factors. Furthermore, biochemical studies of TFIID-TATA box interactions have indicated, first, that both the association and dissociation rates are slow and, second, that efficient binding requires higher temperatures (Nakajima et al., 1988; Horikoshi et al., 1988b; Schmidt et al., 1989a). These characteristics of TFIID are different from those of many other sequencespecific DNA binding factors, which generally can bind DNA rapidly and at low temperatures (Horikoshi et al., 1988b). We (Horikoshi et al., 1989b) and others (Hahn et al., 1989b; Eisenmann et al., 1989; Schmidt et al., 1989b; Cavallini et al., 1989) have recently described the cloning and sequence of the Saccharomyces cerevisiae TFIID gene. Analysis of the TFIID protein sequence failed to reveal any structural motifs previously shown to be involved in DNA binding, such as the helix-turn-helix, Zn*+ finger, or the leucine zipper (Johnson and McKnight, 1989). However, we did note two potentially interesting structural motifs (Horikoshi et al., 1989b): a highly basic domain (residues 120-156) that has the ability to form an a helix and a region (residues 197-240) that bears sequence similarity to the portion of bacterial sigma factors known to interact directly with the -10 element (consensus TATAAT) of bacterial promoters (Helmann and Chamberlin, 1988). Either (or both) of these regions may represent the TATA box binding domain(s) of yeast TFIID. To elucidate the structural basis for the unique properties of TFIID and to test the functional significance of the structural motifs described above, a family of mutated forms of TFIID were generated and tested for their DNA binding and basal level transcription activities. Significantly, each activity required the same large region (residues 63-240) of TFIID, containing both the basic repeat

Cell 1172

TATA box binding

basal level transcription

wild type I

I

+

+

.\z-12

I I

I

+

+

A7-33

0

+

+

I

17-57

0

1

I

+

+

14-62

0

I

1

+

+

\7-83

0

17-117

0

\116-240

-

1177-240

(

~227.240

I

1238-240

1

Figure 1. Structures Yeast TFIID

I

7 I

12

4

3

5

6

7

7

8

8

9

10

11

12

13

b

I

I I

of N-Terminal and C-Terminal Deletion Mutants of

The sequence of yeast TFIID with the N-terminus at the left is illustrated schematically. The basic repeat and sigma homology regions (Horikoshi et al., 198913) as well as the interrupted direct repeats (Cavallini et al., 1969) are indicated. The blocks below the model TFIID structure represent the portion of yeast TFIID retained in the deletion mutants, while the deleted portion of the molecule is shown as a gap. The numbers on the left indicate the actual residues deleted. The columns on the right summarize the TATA box binding and basal level transcription activities as determined by the experiments depicted in Figure 2.

1

2

3

4

5

6

9

10 11 12 13 14 15 16

-43

-31

-22

(residues 120-156) and the sigma factor homology (residues 197-240) as well as a recently noted direct repeat (Cavallini et al., 1989). These results indicate that either the domains for these two activities overlap extensively or that the overall tertiary structure of TFIID is rather inflexible. Additional observations indicate that TFIID binds DNA as a monomer. Results Analysis of TFIID N-Terminal and C-Terminal Deletions To roughly localize the region(s) of TFIID necessary for specific TATA box binding and basal level transcription activities, a series of N-terminal and C-terminal deleted forms of TFIID was made as detailed in Experimental Procedures. In brief, deletion mutants were constructed by the method of Kunkel et al. (1987) by hybridizing a single-stranded form of the wild-type TFIID gene with a mutagenic oligonucleotide. Annealing of the mutagenic oligonucleotide simultaneously caused the introduction of both a deletion and a new restriction endonuclease recognition site (which did not cause a change in the coding sequence) into the gene. The new restriction enzyme cleavage site served as a marker for the successful incorporation of the mutation. A summary of the N-terminal and C-terminal deletion mutants so constructed is presented in Figure 1. Also indicated in Figure 1 are the previously noted (Horikoshi et al., 1989b) basic repeat and sigma factor homologous regions of TFIID, as well as the interrupted direct repeats (Cavallini et al., 1989).

-14 12345676

9

10

11

Figure 2. TATA Box Binding and Basal Level Transcription N-Terminal and C-Terminal Deletion Mutants of TFIID

12

Activities of

N- and C-terminal deletion mutants (described in Figure 1) were expressed and analyzed as described in Experimental Procedures. (a) Analysis of TATA box binding activity by gel mobility shift assay. Reactions contained a major late promoter probe and either no protein (lane 1) or reticulocyte lysate programmed with no RNA (lane 2) or with RNA from wild-type (lane 3) or mutant (lanes 4-13) TFIID genes as indicated. (b) Analysis of basal level transcription activity. Transcription from the adenovirus major late promoter was measured in a control HeLa nuclear extract (lane 1) or in a heat-treated (TFIID-deficient) nuclear extract complemented with buffer (lane 2) human TFIID (lane 3) human TFIID plus reticulocyte lysate (lane 4), reticulocyte lysate alone (lane 5) or reticulocyte lysate programmed with RNA transcribed from wildtype (lane 6) or mutant (lanes 7-16) TFIID genes as indicated. The position of accurately initiated transcripts is indicated with an arrow. (c) SDS-PAGE analysis of mutant TFIID polypeptides. In vitro translation reactions contained either no RNA (lane 1) or RNA from wild-type (lane 2) or mutant (lanes 3-12) TFIID genes.

These mutated TFIID genes were transcribed with T7 RNA polymerase, and the resulting RNAs were translated in a rabbit reticulocyte lysate. Analysis of 35S-labeled translation products by denaturing polyacrylamide gel electrophoresis under reducing conditions is shown in Figure 2c. All of the mutated forms of TFIID mRNA had similar template activities (within a factor of 2) (compare signal intensities in Figure 2c, lanes 2-12) and the sizes of the translated polypeptides were roughly proportional to the sizes of the residual TFIID coding sequences within

Structure-Function 1173

Analysis of TFllD

direct

repeals

-basic &L?at -

sigma homology 2w

1w wild type

TATA box . binding I

r

basal level transcrlption

+

+

+

+

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,194.115, !‘.115-134r

r

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.%155-1691

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2.174.181e

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l-----l

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0

3214.223t

IO

1

-227.2401

f?gure 3. Structures Internally deleted scribed in Figure binding and basal periments shown

I

-

of Internal Deletion Mutants of TFIID

forms of TFIID are represented schematically as de1. The columns on the right summarize the TATA box level transcription activities as determined by the exin Figure 2.

the mutated genes (compare Figure 1 and lanes 2-12 in F:igure 2~). The intact and mutant forms of TFIID produced in the reticulocyte lysate were tested for their ability to bind to the adenovirus 2 major late promoter TATA box using the gel shift assay (Figure 2a). Protein synthesized in response to the wild-type RNA generated a DNA-protein complex (lane 3) that was not observed in the absence of RNA (lane 2) and was shown previously (Horikoshi et al., 1989b) to result from specific binding to the TATA box sequence. Mutant forms of TFIID with N-terminal deletions extending from amino acid residue 2 to 62 bound specifically to TATA box DNA in a fashion qualitatively similar to wild-type TFIID (lanes 3-7). Interestingly, removal of a small number of N-terminal amino acid residues actually increased the extent of TATA box binding by TFIID (compare lane 3 with lanes 4-7). In contrast, both larger N-terminal deletions (i.e., past residue 62) and all C-terminal deletions, including one missing only the last three residues, resulted in the production of polypeptides that were unable to bind specifically to TATA box sequences (lanes 8-13). These results suggested that the TATA box binding domain of yeast TFIID resides in the C-terminal portion of the molecule, somewhere between amino acid residues 63 and 240.

The ability of the deletion mutants to support basal level transcription in conjunction with other general initiation factors from HeLa cells is shown in Figure 2b. As indicated, addition of human TFIID to a TFIID-deficient (heat-

treated) nuclear extract restored activity (lanes l-3). Although the presence of the reticulocyte lysate lowered the TFIID activity (lane 4 versus lane 3), the complementation system still showed a specific response to translated wildtype yeast TFIID (lane 6 versus lane 5). Mutations deleting either part or virtually all of the region between residues 2 and 62 had no detectable effect on transcription (lanes 6-IO), whereas N-terminal deletions extending to or past residue 83 eliminated transcription (lanes 11 and 12). As expected from the DNA binding studies, all of the C-terminal deletions, including A238-240, eliminated transcriptional activity (lanes 13-16). That no corresponding increase in basal level transcription was observed with those mutants that showed increased TATA box binding (above) may reflect the use of different assay conditions or, possibly, a function (activity) that is manifested only in the presence of regulatory factors. Altogether, these studies indicated that the TATA box binding and basal level transcription activities might be colocalized in the same long C-terminal region. Analysis of Altered Forms of TFIID Bearing Small Internal Deletions To examine the role of sequences within the C-terminal region (residues 63-240) of TFIID in a more detailed fashion, a systematic series of mutants bearing small deletions was constructed and analyzed as described for the terminal deletion mutants. As summarized in Figure 3, the deletions averaged 16 amino acids in length and were introduced approximately every 17 residues along the protein coding sequence. Plasmids containing deletion mutations were transcribed in vitro with T7 RNA polymerase, and the derived mRNAs were translated in a reticulocyte lysate. Analysis of 35S-labeled proteins by denaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that all of the mutant TFIID mRNAs had roughly equivalent template activities in the reticulocyte lysate (Figure 4~). As shown in the mobility shift assays of Figure 4a, DNA binding was maintained in mutant proteins missing residues 2-12,13-39, or 34-56, whereas it was eliminated by deletion of residues 58-75 or by all internal deletions downstream of residue 75. Along with the data from Figure 2, this maps the N-terminal limit of the DNA binding domain to residues 63-75. In addition, and in agreement with the results from Figure 2, some of the N-terminal mutations (notably A2-12 and A13-39) markedly increased the DNA binding activity above that of the intact protein. Thus, it appears that essentially all of the residues between residues 63 to 75 and the C-terminus comprise the DNA binding domain, whereas the N-terminus could play some regulatory role in binding. As shown in Figure 4b, the internal deletions that maintained DNA binding (A2-12, A13-39, and A34-56) also effected near wild-type levels of basal level transcription whereas all of the others failed to show any transcriptional activity. These data thus support and extend those observed with the larger terminal deletions and indicate either that there is an overlap of the domains for DNA binding and basal level transcription or that the overall

Cell 1174

b

1234567

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i.:.

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.,

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Figure 5. TFIID Binds to the TATA Box as a Monomer

1

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9

RNAs from wild-type and mutant (A4-62) TFIID were translated either separately or together in the reticulocyte lysate and the resulting polypeptides analyzed for TATA box binding activity (a) or by SDSPAGE (b).

10 11 12 13 14 15 16 17 18 19 20

-31

u/.._

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." -22

-14 12

3

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15

76

Figure 4. TATA Box Binding Activity and Basal Level Transcription tivity of Internal Deletion Mutants of TFIID

Ac-

TATA box binding activity (a), basal level transcription activity (b), and SDS-PAGE analysis of mutant TFIID polypeptides (c). The mutant forms of TFIID indicated in Figure 3 were expressed and analyzed as described in Experimental Procedures. The individual panels are labeled as in Figure 2.

structural integrity of most of the protein is required for the function of individual domains. Since the two cysteines in TFllD appeared to delimit specific domains in the large C-terminal region, the possibility of intramolecular disulfide bond formation was considered. However, mutations of Cys78 to Ala decreased but did not eliminate DNA binding and basal level transcription, while mutation of Cys164 had no detectable effect on either activity (data not shown). Native Yeast TFIID Binds the TATA Sequence as a Monomer The functional form of most specific DNA binding proteins is an oligomeric protein (Johnson and McKnight, 1989). These oligomeric proteins are frequently homodimers (or homotetramers), although recently a number of eukaryotic DNA binding proteins have been documented to bind DNA as heterodimers (Johnson and McKnight, 1989). To determine the polymerization state of the native DNA binding form of TFIID, mobility shift assays were performed with mixtures of wild-type and mutated (A4-62)

forms of TFIID (compare Hope and Struhl, 1987). Both forms of TFIID bound DNA specifically but generated protein-DNA complexes with distinctly different and readily resolvable mobilities (see Figure 2). The mRNAs encoding these two forms of TFIID were translated independently or together and the resulting proteins analyzed. If TFIID binds the TATA sequence element as a dimer (or higher multimer), then the reactions containing a mixture of TFIID molecules should produce a hybrid protein-DNA complex with a mobility distinct from those formed by the independently translated proteins (Hope and Struhl, 1987). Shown in Figure 5b are the results of SDS gel analyses of in vitro expressed wild-type and A4-62 forms of TFIID (lanes 2 and 4, respectively) as well as a cotranslated mixture of the two forms of TFIID (Figure 5b, lane 3). These two forms of TFIID were well resolved. Gel shift assays performed with the cotranslated forms of TFIID showed no complexes other than those found when the individually translated mutant and wild-type forms of TFIID were assayed individually (Figure 5a). Since no heterodimerit forms of TFIID-DNA complexes were generated in this analysis, we tentatively conclude that TFIID binds the TATA box as a monomer. Discussion The importance of understanding structure-function relationships in TFIID is underscored by the central role that this factor plays in transcription: mediating both the assembly of RNA polymerase II and other general factors into a functional preinitiation complex and, at least in some cases, the action of transcriptional regulatory factors. This report extends previous descriptions of the primary structure of yeast TFIID by mapping the domain(s) important for site-specific (TATA) binding and for basal (core promoter) transcription in association with the other general factors. Our results, summarized in Figure 6, indicate that both TATA box binding and basal level transcrip-

Structure-Function

Analysis of TFIID

1175

dlrecl

repeat

dlk?C, repeat

basic repeat

t 1

60

Figure 6. Schematic

78 CYS

Structural

120

stgma

164 CYS

and Functional

homology

t 240

180

Domains

in TFIID

The TATA box binding and basal level transcription domain in yeast TFIID is schematically depicted. The basic repeat, direct repeats, and, in yeast TFIID, sigma factor homologous regions are indicated by horizontal lines, while the positions of the two cysteine residues in TFIID are indicated by squares.

tion activities are colocalized to a large C-terminal region of the protein, whereas an N-terminal region is not required for either activity. The significance of these and other studies showing that TFIID binds DNA as a monomer is discussed below. A Large C-Terminal Portion of TFIID Is Required for TATA Binding TFIID contains none of the conserved structural motifs implicated in DNA recognition by other site-specific DNA binding proteins (Horikoshi et al., 1989b). We therefore undertook a systematic analysis of the DNA binding domain with a series of N-terminal, C-terminal, and small internal deletion mutants. Mobility shift assays indicated that a large C-terminal region (approximately the last 180 residues) of TFIID is required in its entirety for site-specific DNA binding. Importantly, this region contains both of the interesting structural motifs noted previously (Horikoshi et al., 1989b), namely, the central basic core (with repeated lysines) and the region with limited homology to the sigma 2.4 region. The importance of these regions for DNA binding is suggested by the detrimental effect of the small internal deletions, (A115134 and A137-152) which are centered on the basic core, and the C-terminal deletion, which removes only the terminal 3 residues. This large C-terminal region also contains the direct repeats recently noted (Cavallini et al., 1989) within the TFIID sequence. The 180-amino-acid DNA binding domain within TFIID is considerably larger than the DNA binding domains, usually 75-100 residues, of other eukaryotic regulatory proteins (Johnson and McKnight, 1989). Presumably this reflects the following: the relatively unique and preeminent role of TFIID in the recruitment and maintenance of RNA polymerase II and other general initiation factors in a functional preinitiation complex (Nakajima.et al., 1988; Van Dyke et al., 1988); interactions with various activators (Sawadogo and Roeder, 1985; Sawadogo, 1988; Horikoshi et al., 1988a, 1988b; Hai et al., 1988; Abmayr et al., 1988; Workman et al., 1988, 1990); and a role in the exclusion of nucleosomes from promoter regions (Workman and Roeder, 1987; Workman et al., 1988, 1990). These considerations suggest that the DNA binding domain may be, of necessity, structurally complex-especially if a tightly

bound TFllD serves to interface the other general factors with each other or with regulatory factors in the face of a generalized repression mechanism (nucleosome assembly). In addition, previous studies (Davison et al., 1983; Sawadogo and Roeder, 1985; Buratowski et al., 1988; Cavallini et al., 1988; Horikoshi et al., 1988b; Nakajima et al., 1988; Van Dyke et al., 1988; Hahn et al., 1989a; Schmidt et al., 1989a) have indicated that TFIID binds in a slow, temperature-dependent manner, forming a highly stable complex, and the present analysis indicates that TFIID binds as a monomer, consistent with its unique role in forming an asymmetric preinitiation complex. Thus, TFIID behaves differently from most DNA binding regulatory factors and may simply require a correspondingly larger domain(s) for site-specific binding and stabilization. Interestingly, the presence of a repeated motif within the DNA binding domain may allow the formal equivalent of DNA recognition by a dimeric structure, but sequence variations within and flanking these repeats, and the structure of the TATA element itself, could still provide the basis for specific asymmetric binding and transcription initiation. One interesting model, based in part on considerations of bacterial promoter recognition by specific domains in the bacterial RNA polymerase-sigma factor complex (Helmann and Chamberlin, 1988), is that the sigma homology region of TFIID might be responsible for TATA box binding specificity, with the other protein domains (e.g., direct repeats and/or central basic core) involved mainly in stabilizing interactions that are not sequence specific. Previous studies have indicated that nonspecific sequences flanking the TATA element may contribute to the overall binding of TFIID (Sawadogo and Roeder, 1985; Nakajima et al., 1988; Hahn et al., 1989a). Colocalization of the TATA Binding and Transcriptional Activation Domains Given the ability of cloned and expressed yeast TFIID to mediate core promoter function in conjunction with other (human) components of the basic transcriptional machinery, attempts were made to localize regions required exclusively for these interactions and not for DNA binding per se. However, for all the mutants examined, including the small internal deletions, there was complete coincidence between DNA binding and transcription activity. These results suggest either that the activation domain is comprised of all or part of the DNA binding domain or that the overall structural integrity of the large C-terminal portion (residues 83-240) of TFIID is somehow important for the function of distinct individual domains. Relevant to the latter possibility is the observation that while many eukaryotic regulatory proteins are comprised of readily distinguished DNA binding and activation domains (Ptashne, 1988; Mitchell and Tjian, 1989) some do have overlapping or closely interdigitated binding and activation domains that can only be distinguished by refined mutagenesis (Ptashne, 1986; Schena et al., 1989). The same may be expected for TFIID in light of its rather small size and its involvement in a number of interactions, on the promoter, with both general and gene-specific factors.

Cell 1176

If tight binding of TFIID is a prerequisite for promoter activation, then it is easy to understand the failure of DNA binding-defective forms of TFIID to activate transcription. However, it will be important to determine whether the tight binding function of TFIID is required for promoters that have no discernible TATA elements or whether there can be a (partial) compensation for the loss of this function in the presence of regulatory factors that act to facilitate and/or stabilize TFIID binding. Relevant to this, a human TFIID activity appears to be required for transcription of at least one promoter lacking a conventional TATA element (Carcamo et al., 1989), although tests of mutated TATA factors on TATA-less promoters or in response to upstream activators are hampered by the apparent failure of the yeast TFIID to function in these responses. The recent cloning of the human TATA factor cDNA (A. Hoffmann, E. Sinn, T. Y., J. Wang, A. Roy, M. H., and R. G. R., unpublished data) should make such studies possible. Our conclusion that the yeast TFIID contains a C-terminal region important for both DNA binding and basal transcription and a distinct N-terminal region that is not important for these functions is supported by and highly relevant to comparative studies of TFIID structure in other organisms. Thus, analyses of cDNA clones from Schizosaccharomyces pombe (A. Hoffmann, M. H., C. K. Wang, S. Schroeder, l? A. W., and R. G. R., unpublished data), Arabidopsis(A. Gasch, A. Hoffmann, M. H., R. G. R., and N.-H. Chua, unpublished data), Drosophila (M. Muhich, K. lida, M. H., R. G. R., and C. Parker, unpublished data), and humans have revealed that the region corresponding to residues 81-240 in S. cerevisiae is highly conserved (greater than 80% sequence identity), whereas the N-terminal regions are completely divergent. These conclusions also provide a molecular explanation for the ability of just the C-terminal part of yeast TFIID to functionally substitute for the human TFIID in mediating basal promoter activation. Possible Role of the N-Terminal Region of TFIID The present results show clearly that the N-terminal 80-70 residues are dispensable for DNA binding and for basal level transcription in vitro. Although presently there is no direct evidence for any function for this region, it is tempting to speculate that it could be involved somehow in regulatory factor interactions. This proposal is compatible with the failure of yeast TFIID to substitute for human TiIID in mediating the in vitro activity of some mammalian regulatory factors (unpublished data), especially in light of the sequence divergence in the N-terminal regions of the corresponding factors. Of potential interest in this regard is our observation that DNA binding by TFIID is substantially increased by deletions in the N-terminus, suggesting that the natural conformation of TFIID is not optimal for DNA binding and that the N-terminus could be a target for some regulatory factors. These hypotheses must be tested in more defined and sensitive assay systems and under conditions where the action of regulatory factors is apparent.

Experimental Procedures Construction of TFIID Mutants General methods for DNA manipulations were as described (Sambrook et al., 1989). Oligonucleotide-directed mutagenesis was carried out by the methods of Kunkel et al. (1987) to create various TFIID mutants. The original plasmid containing the yeast TFIID gene YCpPD (Horikoshi et al., 1989b) was digested with Accl, repaired with the Klenow fragment of DNA polymerase I, and then digested with Spei. The resulting 837 bp DNA fragment, which contains the entirety of the yeast TFIID coding region and 100 bp of 5’ flanking sequences, was ligated into a pGEM7Zf(+) (Promega) vector that had been digested with Xbal and Smal. The resulting plasmid was transfected into Escherichia coli strain EW313 (dut-, ung), and single-stranded DNA (minus strand) was prepared after superinfection of the cells with helper phage M13KO7. The mutagenic oligonucleotide AGAGAAACTTTTTTAGTATGGCCGATGAGGA (the altered bases are underlined) was treated with T4 polynucleotide kinase and annealed in excess to the single-stranded DNA to create a new restriction enzyme site (Ndel) at the translation initiation region. This mixture was then treated with Sequenase version 2.0 (T7 DNA polymerase) to elongate DNA from the mutagenic primer, and the products were ligated with T4 DNA ligase. The resulting DNA was transfected into E. coli strain DH5a (dut+, ung’) cells to select for the mutated plasmid. Finally, plasmids were digested with Ndel to identify the DNAs that had incorporated the mutagenic oligonucleotide. The resultant plasmids can be used both for in vitro transcription-translation in a reticulocyte lysate (Promega) and an E. coli T7 expression system. Both N-terminal and internal deletion mutants of TFIID were made by deletion of the amino acids (DNA sequence) indicated (see Figures 1 and 3) with different mutagenic oligonucleotides. In the case of A2-12, the mutagenized DNA was further digested with Ndel and self-ligated to create the A2-12 mutant. C-terminal deletion mutants were made by creating termination codons at the indicated positions (see Figure 1) with mutagenic oligonucleotides. Mutations in the two TFIID cysteine residues were made by creating an alanine codon at the indicated positions with different mutagenic oligonucleotides (see Table 1). The oligonucleotides used to create all of these mutants are listed in Table 1. In Vitro Transcription and Translation Plasmids containing the TFIID gene were transcribed in vitro with 20 U of T7 RNA polymerase. Following purification by phenol-chloroform extraction and ethanol precipitation, 1 ug amounts of RNA were used to program in vitro translation reactions (final volume = 25 ~1) containing rabbit reticulocyte lysate (17.5 ~1) according to conditions specified by the supplier (Promega). After incubation, glycerol was added to a final concentration of 10%. Aliquots were employed directly for gel mobility shift and in vitro transcription assays (below). For quantitative analysis of translated proteins, reaction also contained [%]methionine (20 PCi, 1200 Cilmmol), and aliquots (OS ~1) were added to 20 PI of SDS gel sample buffer, heated at 100°C for 2 min, and analyzed in 12% (w/v) polyacrylamide gels containing SDS. Gels were fixed, stained with Coomassie brilliant blue R-250, and exposed to X-ray film. Gel Mobility Shift and Transcription Assays For gel mobility shift assays, 0.5 ul aliquots of reticulocyte lysateexpressed TFIID were incubated with an end-labeled DNA probe under previously described conditions (Horikoshi et al., 1989a. 1989b) that included 100 ng of poly(dG-dC) carrier. The 184 bp probe containing the adenovirus major late promoter (position -138 lo +46) was prepared and corresponding DNA-protein complexes analyzed as previously described (Horikoshi et al., 1989a, 1989b). Transcription assays used untreated HeLa cell nuclear extracts or heat-treated (15 min at 47%) extracts lacking endogenous TFIID (Nakajima et al., 1988), an intact plasmid template (pMLH1) containing the adenovirus major late promoter, and either partially purified human TFIID or aliquots (6 ~1) of translation reactions containing expressed yeast TFIID (Horikoshi et al., 1989b). Specifically initiated adenovirus transcripts were monitored by reverse transcriptase extension of a 30-nucleotide primer complementary to nucleotides +25 to +54 (Horikoshi et al., 1989b).

Structure-Function 1177

Analysis of TFIID

Table 1. Oligonucleotides

Used to Create TFIID Mutants

Mutant

Oligonucleotide

A2-12 A7-33 A7-57 A4-62 67-03 A7-117 A 116-240 A 177-240 A227-240 A236-240 A 13-39 A34-56 A56-75 A75-94 A94-115 A115-134 A137-152 A155-169 A174-181 Ala&195 4 197-208 4214-223 lCYS78 1Cysl64

CGT CAT ATG TTT CAT ATG AAA CGT GAA CCT CGT AAC GAA CAA CAT GAG GAC GCT TTC TTC TTT GTT GCA ATA

The newly created

restriction

TTA ATG GCC TTA ATG GCC ACT CTA ATT GTG TTA CAG TCT ACC GCC CCA TCA GCT CCT TCC CCT TCA ACT CAA

AAG GCC GAT CAT GCC GAT ACA GAA TAC CTA AAG AAT GAA ATT CGT AAA AAG AAA ATA TCC GGT GGA GTG AAT

enzyme

GAG GAT GAG ATG GAT GAG GCT GGG CAA AGT GAG CGA AAA GTG AAT ACT CTG TTC CGT TAT TTG AAG ACT ATT

TTT GAG GAA GCC GAG GAA TTA TTA GCT GAA TTT GAT GAC GCA GCA ACA GCC ACA CTA GAG ATC ATT TTG GTC

sites are indicated.

CAT GAA CGA GAT GAA CGA ATT GCA TGA TTC AAA GGT ACC ACT GAA GCC AGT GAT GAA CCA TAT GTA GGG GGG

ATG AGG TCG ATC AGG TCG TGA TGA TAT TGA GAA ACC TTG GTC TTC TTG ACT ATC TTC AGG CGA TAC GCC TCC

The sequence

Acknowledgments We appreciate the helpful advice of Drs. Shona Murphy and Hildegard Kaulen concerning in vitro mutagenesis and thank Dr. Eric Sinn for reading the manuscript, M. H. is an Alexandrine and Alexander L. Sins,heimer Scholar. This study was supported by grants from the National IInstitutes of Health to P A. W. and R. G. R. and by 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 be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Fleceived March 13, 1990; revised April 23, 1990. References Abmayr, S. M., Workman, J. L., and Roeder, R. G. (1988). The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID : promoter interactions. Genes Dev. 2, 542-553. Breathnach, R., and Chambon, f? (1981). Organization and expression of eucaryotrc split genes coding for proteins Annu. Rev. Biochem. 50, 349-383. Buratowski, S., Hahn, S., Sharp, P A., and Guarente, L. (1988). Function of a yeast TATA element-binding protein in a mammalian transcription system. Nature 334, 37-42. Buratowski, S., Hahn, S., Guarente, L., and Sharp, f? A. (1989). Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549-561. Carcamo, J., Lobos, S., Merino, A., Buckbinder, L., Natarajan, V., and Reinberg, D. (1989). Factors involved scription by mammalian RNA polymerase II: role of MLTF in transcription from the adenovirus major promoters. J. Biol. Chem. 264, 7704-77l4.

GCA ACC GCC GTT ACC GGG TAT TAT CCT AAA TTC TCC GGG AAC GCC GCC GAC CGT TCC ATG TCG CAA CGT GCT

Weinmann, R., in specific tranfactors IID and late and IVa2

Cavallini. B., Huet, J., Plassat, J.-L., Sentenac, A., Egly, J.-M., and Chambon, F? (1988). A yeast activity can substitute for the HeLa cell TATA box factor. Nature 334, 77-80.

AAC AAA ACA CCA GTT AAA CCA CAT ATA ATG CAG GCC TGC CCC TCA AGT TTC CTA TCC GTG GGA GCT TTA GAC

AAG CCA TCA ACA GCG ATG GGG GGT TAC TGA AGT ACA AGG AAG GGG AGA AAA GAA TAT AAG AAG TTT GAT GTT

ATA GCA GGT CTA CTA GTT AAA ACT CCT TGG GAA TCA TTA CGT AAA AAA ATA GGG GAG CCG ATT GAA CTG AAA

GTG ACT ATT CAA CAT GTT ATG TTC GTC GGA GAG GGT GAT TTT ATG TAT CAA TTA CCA AAA GTT GCT AAA TTC

of all mutants was confirmed

(Ndel) ACT GTT AAC GCC ACC GTT TCC CTA (EcoRI) GAC (Kpnl) (Styl) GCT GTT GCA AAT GCA (EcoRI) ATT CTT (Accl) ACA CCT

TTC (Pvul) (ECORV) CGT (Pvul) GTT TCC (EcoRV)

(Avall)

(Avall) (EcoRV) (EcoRV)

(EcoRI)

(Hincll) (EcoRI) (Styl) (Seal) (EcoRV) (StYI) ACT

(Pvul)

GTT ATA

(44 (Avall)

by nucleotide

sequence

analysis.

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