Cell, vol. 64,

971-961,

March 6,

1991,

Copyright 0

1991 by Cell Press

Mechanism of Action of an Acidic Transcriptional Activator In Vitro Young-Sun Lin’ and Michael Ft. Green’ Department of Biochemistry and Molecular Harvard University Cambridge, Massachusetts 02138

Biology

Summary Transcription of a eukaryotic structural gene by RNA polymerase II requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Here we analyze affinity-purified complexes at various stages of assembly to determine the mechanism of action of an acidic transcriptional activator. We show that the activator can function in the absence of ATP and stimulates transcription by increasing the number of functional preinitiation complexes. The activator effects this increase by recruiting the general transcription factor TFIIB to the promoter. Using protein affinity chromatography we demonstrate a specific interaction between an acidic activating region and TFIIB. Based on these combined results, we propose that TFIIB is a direct target of an acidic activator. Introduction Factors involved in the accurate transcription of eukaryotic structural genes by RNA polymerase II can be classified intotwo groups. First, general or basic transcription factors are necessary and can be sufficient for accurate transcription initiation (reviewed in Saltzman and Weinmann, 1989; Lillie and Green, 1989). These basic factors include RNA polymerase II itself, the TATA box-binding protein (TFIID), and several other chromatographically defined factors including TFIIB and TFIIEIF. Another factor, designated TFIIA, has also been implicated as a general transcription factor, although the requirement for it has been highly variable (Sawadogo and Roeder, 1985; Egly et al., 1984; Samuels and Sharp, 1986; Buratowski et al., 1989; Reinberg et al., 1987; Van Dyke et al., 1989; Sumimoto et al., 1990). The general transcription factors assemble on the promoter in an ordered fashion to form a preinitiation complex (see Reinberg et al., 1987; Reinberg and Roeder, 1987; Buratowski et al., 1989, and references therein). Transcriptional activity is greatly stimulated by the second class of factors, promoter-specific activator proteins (activators). In general, cellular activators are sequencespecific DNA-binding proteins whose sites are present within the vicinity of their target promoters (reviewed in Ptashne, 1988; Mitchell and Tjian, 1989; Ptashne and Gann, 1990). How do activators stimulate transcription? lt iS pmsumed that activators interact with or modulate the activity ‘Present address: Program sachusetts Medical Center,

in Molecular Worcester,

Medicine, University of Massachusetts 01605.

MaS-

of one or more of the basic transcription factors (reviewed in Lewin, 1990; Ptashne and Gann, 1990). This supposition raises two questions. First, which of the general transcription factors is the direct target of the activator? Various studies have provided conflicting conclusions. For example, it has been proposed that the direct target of the activator is TFIID (Horikoshi et al., 1988a, 1988b; Stringer et al., 1990), an “intermediary” protein perhaps associated with TFIID (Pugh and Tjian, 1990; Berger et al., 1990; Kelleher et al., 1990), and RNA polymerase II (Allison and Ingles, 1989). A second question is how does the interaction between the activator and its target stimulate transcription? Prokaryotic activators can act at different steps along the pathway leading to transcription initiation (reviewed. in McClure, 1985). Some prokaryotic activators, such as the Escherichia coli OmpR protein (Tsung et al., 1990), stimulate transcription solely by increasing the affinity of E. coli RNA polymerase for promoter DNA. In contrast, h repressor and Salmonella typhimurium NtrC proteins act subsequent to binding of RNA polymerase by increasing the rate of isomerization of the “closed” to the “open” promoter complex. h Repressor and S. typhimurium NtrC proteins differ, however, with regard to their biochemical requirements: NtrC requires ATP for function (Popham et al., 1989), whereas h repressor does not. An activator could also function following transcription initiation. For example, it has been proposed that the HIV-1 Tat protein (reviewed in Cullen, 1990) and the Drosophila heat shock transcription factor (Rougvie and Lis, 1988) act by overcoming a block to RNA polymerase II elongation. We have previously described an in vitro system for analyzing the function of a eukaryotic acidic transcriptional activator (Lin et al., 1988). GAL4-AH (previously referred to as GAL4(1-147)+AH; Lin et al., 1988), a derivative of the yeast activator GAL4, is synthesized in and purified from E. coli. The E. coli-derived GAL4-AH efficientlystimulates transcription when added to a HeLa cell nuclear extract, which lacks endogenous GAL4 DNA-binding activity. Here we use this system, in conjunction with methods to purify functional preinitiation complexes, to determine the step at which an acidic activator functions. We then perform protein affinity chromatography experiments to identify the target of an acidic activating region. Results Experimental Design To study the relationship between preinitiation complex assembly and transcription, we have analyzed affinitypurified complexes formed under different conditions and at various stages of assembly. Our purification method is a modified version of that described by Arias and Dynan (1989). A DNA fragment containing GALCbinding sites upstream of the adenovirus E4 TATA box was attached at one end to streptavidin-agarose beads via a biotin moiety.

Cell 972

GAL4:

- +

--++

-1

E4

lr Hexoklnase: GAL4:

-

I

-

-

aB -

- +

+ +

1

Incubation

Wash: GAL4:

-

t

+

4

I

+

+

E4

+

1

Incubation

++++ -+-+

-

-

+

Wash:

-

+

-

+

- +

-

+

NTPs Incubation

NTPs Incubation

t

RNA Analysis

1

RNA Analysis

+ Figure 2. Function Figure 1. Evidence Assembly

that GAL4-AH

Facilitates

Preinitiation

Complex

Transcription was performed directly in the crude nuclear extract. The diagram of the experimental protocol is shown below the autoradiogram. Transcripts were quantitated by primer extension, and the position of accurately initiated E4 transcripts is indicated on the right. The presence (+) or absence (-) of GAL4-AH in the initial reaction mixture or following washing of the immobilized DNA templates is indicated below each lane.

The DNA beads were then incubated in a HeLa cell nuclear extract, or subsets of partially purified general transcription factors, in the presence or absence of E. coli-derived GAL4-AH (Lin et al., 1988). Factors stably associated with the DNA template are readily separated from free factors by washing the streptavidin-agarose beads in transcription buffer (Arias and Dynan, 1989; see below). Transcription is initiated from these purified, immobilized preinitiation complexes by addition of ribonucleoside triphosphates (NTPs), and the transcripts synthesized are quantitated by primer extension (Lin et al., 1988). In control experiments (data not shown; see below) we found that stimulation of transcription by GAL4-AH was roughly comparable on free and immobilized DNA templates. Furthermore, transcription from immobilized templates was inhibited by 1 ug/ml a-amanitin, indicating that the transcripts are the products of RNA polymerase II (data not shown). GAL4-AH Does Not Increase the Transcriptional Activity of Preassembled Complexes To test whether GAL4-AH acts before or after preinitiation complex assembly, we first performed the experiment shown in Figure 1. We investigated whether transcription from a preinitiation complex, assembled and purified in the absence (or presence) of an activator, could be stimulated by the subsequent addition of an activator. Preinitiation complexes were purified from crude reaction mixtures that contained or lacked GAL4-AH. GAL4AH was then added to the purified complexes and transcriptional activity measured. The results of Figure 1 show that GAL4-AH could not increase transcription from a puri-

of GAL4-AH

Does

Not Require

ATP

The diagram of the experimental protocol is shown below the autoradiogram. The presence (+) or absence (- ) of GAL4-AH and hexokinase in the initial reaction mixture is indicated below each lane.

fied preinitiation complex originally formed in either the absence or presence of GAL4-AH. In contrast, GAL4-AH markedly stimulated transcription if free general transcription factors (nuclear extract) were present. These results suggest that GAL4-AH does not stimulate transcription by increasing the activity of a preassembled complex, but rather by facilitating preinitiation complex assembly. Further support for this conclusion is provided below. GAL4-AH Can Act in the Absence of ATP Initiation of transcription involves one or more ATP-dependent steps (Bunick et al., 1982). This prompted us to test whether ATP hydrolysis was required for the function of the acidic activator. The results of the above experiments, in which preinitiation complexes were formed and purified in the absence of added NTPs, suggested that GAL4-AH action was ATP independent. It remained possible, however, that in these experiments the nuclear extract contained a low level of residual ATP required for GAL4AH to function. To rule out this possibility, we pretreated the nuclear extract with hexokinase and glucose to ensure that all ATP was depleted (see, for example, Bieker et al., 1985; Arias and Dynan, 1989). Purification of preinitiation complexes from these ATP-depleted extracts also removes the hexokinase, enabling transcription to occur following addition of NTPs. Figure 2 shows that the activities of purified preinitiation complexes assembled in the presence or absence of hexokinase were equivalent (compare lanes 4 and 8). Since GAL4-AH can function only if free general transcription factors are present (Figure l), we conclude that GAL4-AH has acted prior to the purification of preinitiation complexes and thus in the absence of ATP. Because accurate transcription initiation involves one or more ATP-dependent steps (Bunick et al., 1982) we can further conclude that GAL4-AH acts prior to the formation of the first phosphodiester bond.

Cdl 974

B Factors:

I)1I# GAL4:

E4

--

-+-+

GAL4:

-+-+-+

incubation Wash

Incubation Wash GAL4:

+ I +

I + I

GAL4:

+ I + m

NTPs RNA Analysis

NTPs RNA Analysis 4. Recruitment

of Factors

to the Preinitiation

Complex

+ I + I + I Missing Factors Incubation

Missing Factors Incubation

Figure

-+-+-+

by GAL4-AH

(A) Recruitment assay. General transcription factors present in the first reaction mixture are indicated above the lanes, and the presence (+) or absence (-) of GAL4-AH in the first reaction mixture is indicated below the lanes. The diagram of the experimental protocol is shown below the autoradiogram. (B) Recruitment of TFIIB requires bound TFIID. Experiment performed as in (A).

tectably increase the amount of TFIID stably associated with the DNA template: similar transcription levels were observed when the first reaction mixture contained either TFIID alone, or GAL4-AH plus TFIID. Comparable results were obtained using a 5-fold lower amount of TFIID (data not shown). When the first reaction mixture contained TFIID and TFIIB, the addition of GAL4-AH significantly increased transcription. This indicates that GAL4-AH had recruited TFIIB into the complex. The control experiment in Figure 46 shows that recruitment of TFIIB requires TFIID: when the first reaction mixture contained TFIIB but not TFIID, transcription was undetectable even when GAL4-AH was present. Thus, in the absence of TFIID, TFIIB does not stably associate w.ith the DNA template, a conclusion in agreement with other studies (Reinberg and Roeder, 1987; Buratowski et al., 1989; Sumimoto et al., 1990; Maldonado et al., 1990). Figure 4A also shows that GAL4-AH can recruit factors that enter the preinitiation complex after TFIIB, but only if TFIIB is present. If all general transcription factors were present in the first reaction mixture, GAL4-AH had a large effect (lanes 9 and 10). Thus, GAL4-AH had promoted the assembly of factors, including RNA polymerase II and TFIIEIF, into preinitiation complexes. But in the absence of TFIIB, RNA polymerase II and TFllElF did not stably associate with DNA even if GAL4-AH was present (lanes 6 and 8). (Note that the low, equal level of transcription in

lanes 7 and 8 was due to contaminating TFllElF activity present in one or more of the factors added in the second reaction mixture; see Figure 8A.) These data are consistent with the hierarchichal order of general transcription factor assembly proposed by Buratowski et al. (1989). On the basis of the results in Figures 3 and 4, we conclude that GAL4-AH can recruit TFIIB to a DNA template containing bound TFIID. Assembly of TFIIB in turn enables RNA polymerase II and TFllElF to enter the complex. Interaction of a General Transcription Factor with a Potent Acidic Activating Region Although the experiments described above implicated TFIIB in the activation process, they did not reveal which factor was directly contacted by the activator. In particular, the ability of GAL4-AH to enhance TFIIB binding required that TFIID be bound to the promoter. These results were consistent with either TFIID or TFIIB being the target of the activator. We therefore initiated a series of protein affinity chromatography experiments to identify the factor that directly interacts with an acidic activation region. Previous studies have shown that the HSV-1 VP16 protein contains a carboxy-terminal acidic activating region that is particularly potent both in vivo (Triezenberg et al., 1988; Sadowski et al., 1988; Carey et al., 1990a) and in vitro (Chasman et al., 1989; Carey et al., lQQOa, 1990b; Berger et al., 1990). We reasoned that the strength of the VP16 activating region would facilitate detection of

Mechanism 973

of Action

1st Incubation

of GAL4-AH

In Vitro

NE: +++++++ GALa: 1+11111 [ I Wash: I - ++++ -0

+ oo]E4

+++++ =+++ --++,,=+-I ,,I,+

2nd Incubation

-

NTPs Incubation 1 RNA Analysis 1 Figure 3. Association Complex Assembly

of TFIIS

Is a Rate-Limiting

Step in Preinitiation

The experimental protocol is diagrammed; the factors present in the first and second incubations are shown above and below the autoradiogram, respectively. The autoradiogram is placed after the first incubation for ease of presentation.

Assembly of TFIIB Is a Rate-Limiting Step in Formation of the Preinitiation Complex The results of the above experiments strongly suggested that GAL4-AH promotes preinitiation complex assembly. This implies that in the absence of GAL4-AH, preinitiation complex assembly stalls at a rate-limiting step. To identify this step, we performed the experiment shown in Figure 3. Complexes assembled in the absence of an activator were purified and then complemented with GAL4-AH and various general transcription factors to determine how far assembly had proceeded. Standard procedures were used to fractionate the crude nuclear extract into the following partially purified components: TFIIA, TFIIB, TFIID, TFIIEIF, and RNA polymerase II (see Experimental Procedures). The activity of these fractions in reaction mixtures containing GAL4-AH is shown in Figure 6A. The fully activated level of transcription was observed upon mixing of all fractions. Transcription was undetectable when either the TFIID, TFIIB, or RNA polymerase II fraction was omitted. Upon omission of the TFllElF fraction, a low level of transcription could be detected, indicating that one or more of the other fractions contained aTFllE/F contamination. (The TFllElF contamination was only evident when transcription was increased to high levels by addition of GAL4-AH; data not shown.) We did not observe more than a modest stimulation by adding the TFIIA fraction, a result in agreement with several previous reports (see, for example, Sawadogo and Roeder, 1985; Horikoshi et al., 1988a, 1988b; Hai et al., 1988; Van Dyke et al., 1988,1989; Sumimoto et al., 1990). We therefore did not include the TFIIAfraction in the experiments described below.

The immobilized DNA templates were incubated in a crude nuclear extract in the absence of GAL4-AH, and complexes were purified by washing. GAL4-AH was added to these purified complexes along with subsets of the partially purified general transcription factors, selected based on their known order of assembly (Reinberg et al., 1987; Reinberg and Roeder, 1987; Buratowski et al., 1989; see Figure 10). Following a second incubation, transcriptional activity was measured. Addition of only GAL4-AH to these purified complexes did not increase transcription above the basal level. Transcription was also not increased by addition of GAL4-AH and TFIIEIF, the final factor to be assembled into the preinitiation complex, or by addition of GAL4-AH, TFIIEIF, and RNA polymerase II. However, transcription was dramatically increased to the activated level by addition of GAL4-AH, TFIIE/F, RNA polymerase II, and TFIIB. Addition of all three general transcription factors (TFIIE/F, RNA polymerase II, and TFIIB) was required to achieve the activated transcription level (data not shown). Because addition of TFIID was not required, we conclude that TFIID was already bound to the DNA template. In support of this conclusion, addition of a heat-treated nuclear extract, which lacks TFIID activity (Nakajima et al., 1988; Pugh and Tjian, 1990; data not shown), restored transcription to the activated level. Taken together, these results indicate that in the absence of an activator, complex assembly stalls following TFIID binding. Therefore, the next event, assembly of TFIIB into the complex, is a rate-limiting step. The experiments presented below confirm and extend this conclusion. GAL4-AH Recruits TFIIB to the DNA Template One way by which GAL4-AH could function is to accelerate a specific step in preinitiation complex assembly. For example, GAL4-AH could assist one (or more) of the general transcription factors to assemble into the complex. To test this possibility, we designed an assay that measures “recruitment” of general transcription factors by GAL4-AH. Reaction mixtures were assembled that contained subsets of the general transcription factors, chosen based on the proposed order of preinitiation complex assembly (Reinberg et al., 1987; Reinberg and Roeder, 1987; Buratowski et al., 1989; see Figure 10). Following incubation, the immobilized DNA templates were washed, and all factors absent from the first reaction mixture were added along with NTPs. Thus, in each instance, GAL4-AH and all general transcription factors were added, but their order of addition was varied. If all factors in the first reaction mixture stably associate with the DNA template, transcription will occur upon addition of the missing factors. In contrast, if any factor in the first reaction mixture does not stably associate with the DNA template, it will be absent from the final reaction mixture and transcription will not occur. We are thus testing whether in the first reaction mixture GAL4-AH can recruit a factor to the complex and, if so, which factor. Figure 4A shows that when present alone TFIID stably associated with the DNA template: a high level of transcription was obtained when the first reaction mixture contained only TFIID. Furthermore, GAL4-AH did not de-

Mechanism 975

of Action

of GAL4-AH

In Vitro

A VP16ColumnEluate: GAL4:

-----II

,,,+-+-+-+I+ I+++++++++++

----II GAL4:

-+

-+

-+

-+

@I

E4

Transcripts

B GST-VP16 Figure 5. A General Activating Region

Transcription

Factor(s)

interacts

with the VP16

200mM KCI Flowthrough +

A HeLa cell nuclear extract (10 ml) was chromatographed in 200 mM KCI on 5 ml of a protein affinity column as described in Experimental Procedures. The flowthrough fraction (25 VI), the eluate (10 PI), or a mixture of the flowthrough and eluate was tested for transcriptional activity in the absence (-) or presence (+) of GAL4-AH in a total reaction volume of 40 ~1. Transcripts were quantitated by primer extension. The positions of the primer and accurately initiated E4 transcripts are indicated on the right. A schematic diagram of the DNA template is shown below the autoradiogram. Figure 6. The General Transcription acts with the VP16 Activating Region

potentially weak protein-protein interactions. Furthermore, there are known amino acid substitution mutants within the VP16 activating region that affect transcriptional activity (Cress and Triezenberg, 1990; Berger et al., 1990). We prepared protein affinity columns containing the VP1 6 acidic activating region using the glutathione S-transferase (GST) expression system (Smith and Johnson, 1968). The 78 amino acid carboxy-terminal VP1 6 activating region (Triezenberg et al., 1988; Sadowski et al., 1988) and truncated derivatives of the activating region (see below) were expressed in E. coli as GST fusion proteins and purified on glutathione-agarose beads. We fractionated the HeLa cell crude nuclear extract over a column containing the wild-type 78 amino acid VP16 activating region (GST-VP16). The flowthrough fraction was assayed for its ability to support transcription in the absence or presence of GAL4-AH. Figure 5 shows that when a HeLa cell nuclear extract was chromatographed at 200 mM KCI on the control column containing only the GST protein, the flowthrough fraction supported both basal (- GAL4) and stimulated (+GAL4) transcription at a level comparable to that of the crude nuclear extract. In contrast, when the HeLa cell nuclear extract was chromatographed under identical conditions on the GSTVP1 6 column, the flowthrough fraction, which we estimate contains 99.5% of the total protein, did not support either basal or stimulated transcription. The 1 M KCI eluate from the GST-VP16 column, which represents approximately 0.5% of the total protein, by itself had no transcriptional activity. Transcriptional activity was completely restored upon addition of the 1 M KCI eluate to the GST-VP16 flowthrough. As expected, the 1 M KCI eluate from the GST column did not restore activity to the GST-VP16 flowthrough fraction (data not shown). On the basis of

Factor

TFIIB

Selectively

Inter-

(A) Analysis of the 0.2-l M KCI eluate. Reaction mixtures contained GAL4-AH and combinations of the general transcription factors. The general transcription factor omitted from the reaction mixture is indicated above the lane. The 0.2-l M KCI eluate (10 ~1) from the GSTVP16 column was either added to (+) or omitted from (-) the 40 ~1 reaction mixture. Note that the low level of transcription from reaction mixtures lacking TFllElF was due to minor contamination of TFllElF in one or more of the other fractions. Typically, 3 PI of nuclear extract contains a sufficient amount of each general transcription factor to restore detectable activity to each of these reaction mixtures. The chromatography procedure (see Experimental Procedures) is expected to concentrate bound factors approximately 5-fold. (B) Analysis of the 200 mM KCI flowthrough fraction. Reaction mixtures containing 25 PI of the 200 mM KCI flowthrough from the GST-VP16 column were complemented with the partially purified transcription factor or fraction indicated above the lane in a total reaction volume of 40 PI.

these results, we conclude that one or more general transcription factors interact with the VP16 activating region. TFIIB Is Specifically Retained on the VP16 Affinity Column We performed biochemical complementation experiments to identify the general transcription factor(s) retained on the GST-VP16 column. Five reaction mixtures were assembled containing GAL4-AH and all but one of the general transcription factors. We measured the transcriptional activity of these reaction mixtures in the presence or absence of the 1 M KCI eluate from the GST-VP16 column (Figure 6A). The GST-VP16 column 1 M KCI eluate did not stimulate transcription from reaction mixtures lacking either TFIIEIF, RNA polymerase II, TFIID, or TFIIA. In contrast, the GSTVP1 6 column 1 M KCI eluate dramatically augmented transcription of the reaction mixture lacking TFIIB; the re-

Cdl 976

GAL4: VP16(0.1-1):

‘I

+-+

+

+

obtained with the GST-VP16 column 1 M KCI eluate. Based on these combined results, we conclude that under these conditions TFIIB is the only general transcription factor that interacts with the immobilized VP16 activating region.

+

mm-+-+ 8

-

g]

E4 Transcripts

CIIIIII)

B

GST-VP16

Primer

100mM KCI Flowthrough +

8

-

-

Figure 7. Both TFIIB and TFIID gion at Reduced Ionic Strength

I)]E4

Transcripts

Primer

Interact

with the VP16 Activating

Re-

(A) Analysis of the 0.1-I M KCI eluate. The experiment was as described in the legend to Figure 6A except that the nuclear extract was chromatographed at 100 mM KCI. (6) Analysis of the 0.1 M KCI flowthrough. The experiment was as described in the legend to Figure 66 except that the nuclear extract was chromatographed at 100 mM KCI.

sulting level of transcription was similar to that observed when all general transcription factors were present. The data presented in Figure 6A indicate that under these conditions, TFIIB was the only one of the known general transcription factors that interacted with the VP16 activation region. It is, of course, possible that there are general transcription factors that have not yet been identified and that one or more of these were also retained on the GST-VP16 column. To address this possibility, we performed the reciprocal experiment to that of Figure 6A to determine which of the general transcription factors restored transcriptional activity to the GST-VP16 200 mM KCI flowthrough fraction. Figure 66 shows that, as expected, addition of the GSTVP1 6 1 M KCI eluate to the flowthrough fraction restored transcriptional activity. Addition of either the TFIIA, TFIID, RNA polymerase II, or TFllElF fraction had no significant effect. In contrast, the TFIIB fraction dramatically increased transcriptional activity to a level similar to that

At Reduced Ionic Strength Both TFIIB and TFIID Are Retained on the GST-VP16 Column It has been recently reported (Stringer et al., 1990) that the general transcription factor TFIID binds to a protein affinity column containing the VP16 activating region. However, in our experiments, TFIID activity was in the flowthrough and not the eluate of the GST-VP16 column. We noted several differences between our experimental conditions and those used by Stringer et al. (1990). In particular, we chromatographed the nuclear extract on the affinity column at 200 mM KCI, whereas Stringer et al. (1990) performed chromatography at 100 mM KCI. This prompted us to test whether TFIID would bind to our GST-VP16 column at reduced ionic strength. Figure 7A shows that when the crude nuclear extract WasappliedtotheGST-VPlGcolumnat 1OOmM KCI, both TFIIB and TFIID activities could be detected in the 1 M KCI eluate (approximately 5% of total protein). However, whereas the 1 M KCI eluate completely restored the transcriptional activity of a reaction mixture lacking TFIIB, only partial complementation wasobserved in the reaction mixture lacking TFIID. This suggested that although some TFIID was retained at 100 mM KCI, a significant amount flowed through the column. To confirm that TFIID was present in the flowthrough, we analyzed this fraction by complementation with partially purified general transcription factors. Figure 78 shows that transcription could be detected when TFIIB was added to the 100 mM KCI flowthrough fraction, indicating that TFIID was present. Transcription was not restored to the full level, as expected, since some TFIID was retained on the column (Figure 7A). When TFIID was added to the 100 mM KCI flowthrough fraction, transcription was not detected. Thus, the 100 mM KCI flowthrough fraction lacked at least one factor other than TFIID. Addition of both the TFIIB and TFIID fractions restored transcription to the full level. We conclude that at 100 mM KCI both TFIIB and TFIID interact with the VP16 activating region. However, even under these conditions TFIID was only partially retained, whereas TFIIB was quantitatively retained. We therefore believe that the loss of transcriptional activity following chromatography on a VP1 6 affinity column reported by Stringer et al. (1990) was primarily due to depletion of TFIIB, not TFIID. Partially Purified TFIIB Binds to the VP16 Activating Region In the experiments described above TFIIB was retained on the GST-VP16 column when applied in the presence of the other general transcription factors. It was therefore possible that one of the other general transcription factors facilitated the interaction of TFIIB with the VP1 6 activating region. To address this issue, we tested whether partially purified TFIIB could bind to the VP16 activating region. TFIIB was purified as described by Reinberg and Roeder

Mechanism 977

of Action

E s 8 IL

=

of GAL4-AH

In Vitro

- TFIIB

aII

VP16A456:

3

8

]E4 Transcripts

111+11+1

VP166456-FP442:

1111+11+

1E4 Transcripts -

Primer w

+

Figure 6. Partially Region

Purified

I I I I ( GAL4

GAL4 Sites TFIIB

Interacts

with the VP16 Activating

TFIIB was purified through the single-stranded DNA-cellulose step as described by Reinberg and Roeder (1967). In biochemical complementation experiments this fraction did not contain detectable amounts of any of the other general transcription factors (see text; data not shown). One milliliter of the TFIIB fraction was loaded onto a 1 ml GST-VP16 column preequilibrated with buffer D containing 0.1 M KCI. After the flowthrough fraction was collected, the column was washed extensively with buffer D containing 0.1 M KCI and then eluted stepwise first with buffer D containing 0.2 M KCI and then with buffer D containing 1 M KCI. Each fraction (10 ~1) was used for transcription assays in a total reaction volume of 40 ul.

(1987). In biochemical complementation experiments this fraction did not contain detectable amounts of any of the other general transcription factors (see above; data not shown). The TFIIB fraction was applied to the GST-VP16 column in 100 mM KCI, and the column eluted first with 200 mM KCI and then with 1 M KCI. Each fraction was added to a reaction mixture containing GAL4-AH and all general transcripton factors except TFIIB. As shown in Figure 8, the TFIIB activity was present only in the 1 M KCI eluate, indicating that all of the TFIIB interacted with the VP1 6 activating region in the absence of any other known general transcription factor. A VP16 Single Amino Acid Substitution Mutant Does Not Bind TFIIB The experimentsdescribed above strongly suggested that TFIIB directly interacts with the VP16 activating region. However, because the acidic VP16 activating region is highly negatively charged, it remained possible that retention of TFIIB on the GST-VP16 column was due to a nonspecific ionic interaction rather than a specific proteinprotein interaction. To address this possibility, we analyzed a mutant VP1 6 derivative. VP16A456 bears a truncated 43 amino acid version of the 78 amino acid VP1 6 activating region. VP1 6A456 stimulates transcription in vivo to approximately 400/o-50% of the level of the intact activating region (Triezenberg et al., 1988; Cress and Triezenberg, 1990; Berger et al.,

Figure 9. TFIIB Region

Primer

Does

TATA

r’

Sites

Not

Interact

with

a Mutant

VP16

Activating

Nuclear extract (10 ml) was loaded onto the GST-VP16A456 column or the GST-VP16A456-FP442 column preequilibrated with buffer D containing 0.1 M KCI. Reaction mixtures contained GAL4-AH and combinations of the general transcription factors. The general transcription factor omitted from the reaction mixture is indicated above the lane. The 0.1-l M KCI eluate from the GST-VP166456 column or the GST-VP16A456-FP442 column was either added to (+) or omitted from (-) the reaction mixtures as indicated.

1990). Mutation of phenylalanine 442 to a proline eliminates transcriptional activity in the VP1 6A456 background (VP16A456-FP442) (Cress and Triezenberg, 1990; Berger et al., 1990). We constructed protein affinity columns containing GST-VP16A456 and GST-VP16A456-FP442 fusion proteins. As shown in Figure 9, when the crude nuclear extract was applied to the GST-VP16A456 column, TFIIB was present in the 1 M KCI eluate, indicating that TFIIB bound to the VP16A456 activating region. In contrast, TFIID activity was not detected in the 1 M KCI eluate, indicating that TFIID did not interact with the VP16A.456 activating region. When the crude nuclear extract was chromatographed under identical conditions on the column containing the mutant VP16 activating region (GST-VP16A456-FP442), TFIIB activity was not detected in the 1 M KCI eluate (Figure 9). As expected, all of the TFIIB activity was in the flowthrough fraction (data not shown). Thus, TFIIB interacted with VP16A456 but not VP16A456-FP442. Since the phenylalanine to proline substitution does not alter the overall charge of the protein, we conclude that the association between TFIIB and the VP1 6 acidic activation region results from a specific protein-protein interaction. Discussion Based on the results presented here and previous studies on preinitiation complex assembly (Reinberg et al., 1987; Reinberg and Roeder, 1987; Buratowski et al., 1989, and references therein), we propose a model for howthe acidic

Cell 978

TFIIB binding appears to be rate limiting on the promoter we have analyzed, other promoters may have different rate-limiting steps. The assays described here should provide a means to address these issues.

TATA

+

Figure

10. A Model for How an Acidic Activator

Stimulates

The order of assembly of the general transcription previous studies, in particular that of Buratowski

Transcription

factors is based on et al. (1989).

activator GAL4-AH stimulates transcription in vitro (Figure 10). Initially, the general transcription factor, TFIID, and the activator, GAL4-AH, bind to their respective sites on the DNA template. TFIIB then associates with this ternary complex, and it is this step that is enhanced by the acidic activator. OnceTFllB is bound, theother general transcription factors, RNA polymerase II and TFIIEIF, can stably associate to form a functional preinitiation complex. Thus, by enhancing TFIIB binding, GAL4-AH indirectly recruits RNA polymerase II to.the promoter. Conceivably, TFIIB binding may be enhanced by GAL4AH, but only under specific in vitro conditions or in particular assays. We note, however, that complementary results were obtained when complexes were assembled either in a crude nuclear extract (Figure 3) or using partially purified general transcription factors (Figure 4). Furthermore, in several genomic footprinting studies the TATA box of transcriptionally inactive genes was protected (Wu, 1984; Zinn and Maniatis, 1986; Devaux et al., 1987; Pauli et al., 1987; Albrecht et al., 1988). This is consistent with our in vitro data, indicating that binding of TFIID is not a rate-limiting step. Our results do not exclude that in addition to enhancing the association of TFIIB with the DNA template, GAL4AH functions at a later step in preinitiation complex assembly, or even subsequently; our experimental design only identifies the earliest step affected by the activator. Protein motifs other than acidic regions can serve as activating regions (reviewed in Mitchell and Tjian, 1989; Ptashne and Gann, 1990). Whether these other activators, and different acidic activators, act at the same step as GAL4-AH remains to be determined. Moreover, although

Target of the Activator We have demonstrated a specific interaction between an acidic activating region and TFIIB. Substitution of a single amino acid within the VP16 activating region, phenylalanine 442, can dramatically decrease transcriptional activity (Cress and Triezenberg, 1990; Berger et al., 1990) and TFIIB binding (Figure 9). Thus, binding of TFIIB to the acidic VP1 6 activating region is not the result of a nonspecific ionic interaction. We imagine that phenylalanine 442 either contacts TFIIB directly, or helps structure the surface of VP16 that contacts TFIIB. The relevance of the VPl6-TFIIB interaction is strongly supported by our finding that during preinitiation complex assembly, binding of TFIIB is enhanced by an acidic activator. The model that emerges from these two sets of data is that the acidic activator contacts TFIIB and facilitates its assembly into the preinitiation complex (Figure 10). Several other factors have previously been suggested to be the direct target of a transcriptional activator. Two lines of evidence suggest that TFIID is the target. First, it has been reported that the activators GAL4 (Horikoshi et al., 1988a) and ATF (Horikoshi et al., 1988b) can qualitatively alter the DNAase I footprint of TFIID on the TATA box. The interpretation of these data was that the activators had contacted and induced a conformational change in TFIID. However, the TFIID preparation used in these studies was purified only about 200-fold (Nakajima et al., 1988) and lOO-fold more TFIID per DNA template was used in DNAase I footprinting than in transcription experiments (Horikoshi et al., 1988a, 1988b; Hai et al., 1988). It is therefore possible that the alteration in the DNAase I footprint was due to a factor other than TFIID. In fact, the major alterations in the DNAase I footprint were observed downstream of the initiation site (Horikoshi et al., 1988a, 1988b), a region that appears to be protected by TFIIB in the preinitiation complex (Buratowski et al., 1989; Maldonado et al., 1990). In some promoters the downstream protection is very sensitive to nonspecific DNA competitors, whereas protection over the TATA box is not (Nakajima et al., 1988). It is difficult to understand how these variable sensitivities can result from the same TFIID molecule. Finally, although in vivo footprinting studies reveal protection over the TATA box and activator-binding sites (Wu, 1984; Zinn and Maniatis, 1986; Devaux et al., 1987; Pauli et al., 1987; Albrecht et al., 1988) to our knowledge the extended footprinting pattern has never been observed in vivo. A second argument that TFIID is the target of an activator is that TFIID can bind to a column containing an acidic activating region (Stringer et al., 1990). However, this previous study did not systematically examine whether other general transcription factors, and in particular TFIIB, were also retained on the column. In agreement with the results of Stringer et al. (1990) we found that TFIID could bind to the wild-type VP1 6 protein column at 100 mM KCI (Figure

Mechanism 979

of Action

of GAL44H

In Vitro

7). But the VP16 activating region and its derivatives interact more strongly (or at least in a more salt-resistant fashion) with TFIIB than with TFIID. We were unable to address whether retention of TFIID on the VP16 column reflected a nonspecific ionic interaction or a specific protein-protein interaction: TFIID did not bind to the VP1 6A456 activating region (Figure 9) the appropriate background in which to analyze the phenylalanine 442 substitution mutant. We note, however, that TFIID binds avidly to negatively charged chromatographic resins (reviewed in Saltzman and Weinmann, 1989). It has also been suggested that the direct target of the activator is not a general transcription factor but rather another factor referred to as an “intermediary protein,” “adaptor,” or “coactivator” (Pugh and Tjian, 1990; Berger et al., 1990; Kelleher et al., 1990; reviewed in Lewin, 1990; Ptashne and Gann, 1990). The putative coactivator may be closely associated with one of the general transcription factors and bridge the interaction with the activator (Lewin, 1990; Ptashne and Gann, 1990). If this is the case, the coactivator would have to be present in our most purified TFIIB fraction. Of course, our results do not exclude the possibility that there may be components in addition to the known general transcription factors that are required to achieve the activated level of transcription. Finally, it seems reasonable that an activator, and in particular an acidic activator, may have more than one target. A characteristic feature of eukaryotic activators is their ability to cooperate with one another to stimulate transcription synergistically (see, for example, Carey et al., 1990a; Lin et al., 1990; reviewed in Ptashne, 1988). Transcriptional synergism can be observed with a single activator, when bound at multiple sites on the promoter (Carey et al., 199Oa). Even for a very strong activator, such as GAL4(1147)+VP16, there is a dramatic and synergistic increase in transcription when the number of activator-binding sites is increased from one to two (Carey et al., 1990a). One explanation for this result is that the two bound activators interact simultaneously with a single transcription component (Carey et al., 1990a; Lin et al., 1990). Alternatively, the synergism elicited by two bound GAL4(1-147)+VP16 molecules (Carey et al., 199Oa) may be due to the interaction of one of the bound activators with TFIIB, and the other with TFIID (or another factor). Experimental

Procedures

Plasmid Construction pGVP was cloned by inserting the Hindlll-Bglll fragment of pCRF1 (Triezenberg et al., 1988) containing the carboxy-terminal 78 amino acid VP16 activating region between the EcoRl and BamHl sites of pGEX-PT (Smith and Johnson, 1988). pGVPA456 and pGVPA456FP442 were constructed by replacing the Sphl-BamHI fragment of pGVP with the Sphl-BamHi fragment of pMSVP16A456-490 and pDCFP442(Berger et al., 1990; Cressand Triezenberg, 1990) respectively. immobilized DNA Templates Preparation of immobilized DNA templates was as described by Arias andDynan (1989). pGsE4T(100pg; Linetal., 1988)wascutwith Hindlll and Pvuli, and labeled with Bio-dATP at the Hindlll site with Klenow fragment. The 500 bp DNA fragment containing five copies of GALC binding sites upstream of the adenovirus E4 TATA box was purified on

an agarose gel and coupled to 5 ml of streptavidin-agarose beads and incubated at 4OC overnight. The beads were stored in TE + 0.03% NaN3 at 4%. Under these conditions the beads retained activity as transcription templates for at least 3 months. Preparation of Protein Affinity Columns Overnight cultures of E. coli XA90 transformed with pGEX-PT, pGVP, pGVPA456. and pGVPA456-FP442 were diluted 1:lO in fresh LB medium and grown for 1 hr at 37% before induction with 1 mM IPTG. Three hours after induction, cells were pelleted and resuspended in l/50 culture volume of buffer A (20 mM Tris-HCI [pH 7.41, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) containing 1 M NaCI. Cells were lysed in a French pressure cell or by ultrasonication. Cell debris was removed by centrifugation at 10,000 x g for 10 min. Preswollen glutathione-agarose beads (2 ml per liter of culture) were added to the supernatant. After agitation at room temperature for 5 min the beads were collected by filtration on a sintered glass funnel and washed with 20 vol of buffer A containing 1M NaCl three times and then with buffer A containing 0.1 M NaCl three times. The protein-agarose beads were stored in buffer A containing 0.1 M NaCl and 0.03% sodium azide at 4%. The concentration of the immobilized protein was determined by a Bradford assay using bovine serum albumin as a standard. These concentrations are as follows: GST, 3.8 mg/ml; GST-VP16,2.2 mglml; GST-VP16A456, 2.7 mglml; GST-VP16A456-FP442, 2.6 mglml. Affinity Chromatography Nuclear extract (10 ml) was adjusted to the ionic strength indicated in the figure legends and loaded onto 5 ml of either GST, GST-VP16, GST-VP16A456. or GST-VP168456-FP442 resin preequilibrated with buffer D (Dignam et al., 1983) of the same ionic strength as the applied nuclear extract. The flowthrough was reapplied to the column. After collecting the second flowthrough fraction, the column was washed extensively with the equilibration buffer and eluted with buffer D containing 1 M KCI. The flowthrough (9 ml) and eluate (2 ml) were dialyzed against buffer D containing 100 mM KCI. Purification of Transcription Factors Expression and purification of GALC-AH were as described (Lin et al., 1988). Purification of TFIIA, TFIIB, TFIID, TFIIEIF, and RNA polymerase II was performed essentially as described by Reinberg et al. (1987) and Reinberg and Roeder (1967). Purity was assessed at each step of purification by performing transcription reactions in the presence of GAL4-AH and omitting a single fraction (see, for example, Figure 6A). Purification was taken to the point at which there was no detectable transcription when a single fraction was omitted. As described in the Results, we were unable to completely remove the TFllElF contamination from all other fractions. TFllA The 0.1 M KCI phosphocellulose fraction containing TFIIA activity was loaded onto a DEAE-Sepharose column preequilibrated with buffer D containing 0.1 M KCI. TFIIA was eluted with buffer D containing 0.3 M KCI and dialyzed against buffer D containing 0.1 M KCI. TFlfLI The 1 M KCI phosphoceiluiose fraction containing TFIID was adjusted to 0.1 M KCI and loaded on a DEAE-Sepharose column preequilibrated with buffer D containing 0.1 M KCI. TFIID was eluted with buffer D containing 0.25 M KCI and dialyzed against buffer D containing 0.1 M KCI. TF//B and TFIIEIF TFIIB and TFllElF were prepared from the 0.5 M KCI phosphocellulose fraction by chromatography on a DEAE-Sepharose column preequilibrated with buffer D containing 0.1 M KCI. TFIIB was in the flowthrough, and TFllElF was eiuted with buffer D containing 0.25 M KCl. The TFllB activity present in the flowthrough was further purified on a singlestranded DNA-cellulose column preequilibrated with buffer D containing 0.1 M KCI. TFIIB was eluted from the single-stranded DNAcellulose column with buffer D containing 0.3 M KCI. RNA Polymerase II RNA polymerase II was purifed from nuclear pellets as described by Reinberg and Roeder (1987) through the final phosphocellulose column. The amount of each fraction used in compiementation assays was

Cl?ll 980

determined by titration so that a limiting amount of each fraction was added: reducing either the TFIID, TFIIB, TFIIEIF, or RNA polymerase II fractions decreased transcription (data not shown). The following quantities were used per reaction: 1 pl of TFIIA (0.24 pg), 1 pl of TFllEl F (0.2 pg), 3 pl of RNA polymerase II (0.05 Fg), 5 pl of TFIIB (0.5 Kg), and 5 pl of TFIID (0.23 Kg). In Vitro Transcription Heat treatment of the nuclear extract was as described by Nakajima et al. (1988). Hexokinase treatment of the nuclear extract was performed by incubating 100 nl of nuclear extract, 1 sl of 2.7 M glucose, and 3 U of hexokinase at 30°C for 15 min (see Bieker et al., 1985; Arias and Dynan, 1989). In vitro transcription reactions (40-60 WI) contained either 200 ng of free or 20 pl of immobilized DNA templates. Incubation in the absence of NTPs was performed at 30% for 30 min. Incubation in the presence of NTPs was performed at 30°C for 1 hr. Washing was performed with 1 ml of buffer containing 12 mM HEPES (pH 8.0) 120% glycerol, 60 mM KCI, 0.12 mM EDTA, 7.5 mM MgCl*, and 0.5 mM dithiothreitol three times. All other methods were as described in Lin et al. (1988). Acknowledgments We gratefully acknowledge S. Triezenberg for providing VP16 derivatives and helpful discussions during the course of this work and S. Triezenberg and S. McKnight for the pCRF1 plasmid. We thank K. Yamamoto for insightful suggestions during the course of this work; K. Struhl, A. Hochschild, K. Martin, P. Zamore and M. Zapp, and particularly M. Ptashne for critical comments on the manuscript; and L. Barberis-Maino for superb technical assistance. This work was supported by a grant from the NIH to M. Ft. G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

6, 1990; revised

December

21, 1990

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Mechanism of action of an acidic transcriptional activator in vitro.

Transcription of a eukaryotic structural gene by RNA polymerase II requires the ordered assembly of general transcription factors on the promoter to f...
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