Initiation of transcription by RNA polymerase II

Biochimica et Biophysica Acta, 1009 (1989) 1-10 Elsevier BBAEXP 91992

Review

Initiation of transcription by RNA polymerase 11I F r e d H. M e r r n e l s t e i n , O s v a l d o F l o r e s a n d D a n n y R e i n b e r g Department of Bioch~istry. Robert Wood Johnson Medical School, Unwersity of Medicqne and Dentistry of New Jers~..v, P,'sc~,tawgv NJ (U.S.A.) (Received 30 June 1989) Key words: Transcription initiation: RNA polymerase 1!

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ll.

Structureof class 1! promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11|. Factors operating via common promoter elements, the TATA box and the CAP site . . . . . . . . . . . IV. RNA polymerase II bindingproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Transcriptionof non-TATA-s~quence-co,~¢ainingpromoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Specifictranscription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summaryremarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

L Introduction The initiation of transcfip'~ion is a highly regulated process mediated, in part, by RNA polymerase II. This enzyme catalyzes the de novo synthesis of precursor messenger RNA in vivo. This pro,tess occurs in a 5' --, 3' direction and requires the four ribonucleoside triphosphates for complementary base-pairing as well as a D N A template. Unlike prokaryotic R N A polymerase holoenzyme, purified eukaryotic RNA polymerase II does not recognize promoters in vitro [1]. Furthermore, it transcribes duplex D N A poorly and nonspecifically,

Abbreviations: TF, transcription factor; Ad-MLP, adenovirus major late promoter; RAP, RNA polymerase II associating proteins; ATF, activating transcription factor; MLTP, major late transcription factor; CRE, cAMP response element; PK, protein kinase; TRE, TPA response element; TPA, phorbolester tumor promoter. Correspondence: D. Reinbcrg, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08954-5635, U.S.A.

initiating predominantly at single-stranded regions [1,2]. With the development of cell-free transcription systems [3,4], it was demonstrated that RNA polymerase I| can recognize and accurately initiate transcriptioi~ in vitro at the same sites that are used in vivo [3-6]. This reaction requires the supplementation of additional factors present in crude cell extracts [7-11]. The fractionation and identification of these factors has led to their classification into two basic groups: the general transcription factors, which act at all class I| genes, and a second set of factors termed the specific transcription factors, which operate at unique promoter elements of specific genes. This classification has been supported by the observation that RNA polymerase II and the general transcription factors are required for transcription at all class II promoters. In contrast, specific transcription factors have not been shown to be required for basal levels of transcription, but instead stimulate transcription by enhancing the rate of initiation a n d / o r influence the formation of the preinitiation complex, which is composed of RNA polymerase II and the general transcription factors. This article will review the protein factors

0167-4781/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

that recognize distinct promoter elements and how these factors interact to regulate transcription of defined genes. I!. Structure of class II promoters The promoter elements regulating the transcription of RNA polymerase II transcribed genes (class 11 genes) have been extensively analyzed [12,13]. It has been demonstrated that at least three functionally diverse elements are required for optimal transcription. One such element, the TATA box, in mammalian genes is located approx. 30 nucleotides upstream from the transcription initiation site (CAP site) and positions the start site of transcription [14]. Other elements that serve as recognition sites for specific DNA binding proteins are usually located upstream of the TATA box [12,13,15]. It also appears that there are at least two types of upstream element that may be present in genes. One such type of promoter element can mediate the response to a defined stimulus, such as growth factors, phorbol esters, steroid hormones, heat shock, and heavy metals among others. The second type of upstream element is less specific, and appears to increase the efficiency of expression of a number of genes. Two well-characterized examples include the CAAT box and the SP1 motif [12,13]. A third element regulating transcription is the enhancer, which i~ functionally different from upstream elements. Enhancer elements are defined by their positional independence relative to the CAP site [16]. Our laboratory has characterized some of the factors that operate at these elements which will be summarized here. RNA polymerase II, its structural properties, and the factors that play a role in mediating the RNA polymerase II catalyzed elongation reaction has been recently reviewed by Saltzman and Weinman [17], and will not be discussed in this review. !!!. Factors operating via common promoter elements, the TATA box and CAP site

Five protein factors required for transcription have been defined using the minimal promoter sequence elements, the TATA box and CAP site. The factors operating at these sites were identified and purified by the use of functional transcription assays. These studies have shown that these factors seem to be required for the transcription of all the cla~,s II genes analyzed thus far and therefore have been defined as general transcription factors. Of these five factors, only one has been shown to possess DNA binding activity with specificity for the TATA motif [8,18] and is referred to as TFIID (for alternative terminology, see Table I). The binding of TFIID is considered to be one of the first steps torwards the formation of a DNA protein complex capable of initiating transcription (rapid start-complex) [8,19,20].

The binding of TFIID to the TATA sequence provides the entry of RNA polymerase II into the transcription cycle [19,20]. Even though TFIID has been shown to bind the TATA sequence as demonstrated by DNase I footprint analysis [18,21], binding has not been demonstrated when assayed by gel mobility shift assays. These experiments have suggested that a relatively high dissociation constant (weak binding) exists for TFIID at the TATA sequence. Because of this, the purification of TFIID by DNA affinity chromatography has been unsuccessful. TFIID activity has been purified from Saccharomyces cerevisiae and is capable of functionally replacing the mammalian counterpart in directing transcription from the adenovirus major late promoter (AdMLP) [22,23]. However, molecular weight determinations performed by gel filtration chromatography and glycerol gradient sedimentation has shown that mammalian TFIID activity is approx. 100 kDa [20,24], whereas in yeast [22], and Aspergiilus (Roche, E. and Reinberg, D., unpublished data), this activity has been found at 27 kDa. Greater stability of the yeast factor as compared to the mammalian activity has allowed the purification of TFIID to near homogeneity and will most likely lead to the identification of its sequence. A factor required for transcription and that affects the binding activity of TFIID to the TATA sequence has been identified. This factor has been termed TFIIA. The identity of TFIIA as a bonafide transcription factor is an issue that remains in question. Roeder and coworkers [21,26-28] have been able to reconstitute transcription from at least two different promoters in the absence of TFIIA. It is possible that this finding may have been a consequence of the methodology employed in the isolation of TFIID. These investigators have used heat-inactivated extrP.~.~sas a source for all transcription factors in the purification of TFIID, followed by reconstitution of transcription reactions by the addition of fractions derived from unheated extracts containing active TFIID [18]. It is possible that heat-inactivated extracts also resulted in the inactivation of TFIIA. Therefore, this purification procedure may have resulted in the isolation of a fraction that contains both activities, T F I I D and TFIIA. Furthermore, TFIID and TFIIA have been shown to copurify [20]. The isolation of a partially purified, transcriptionally active fraction of mammalian TFIID containing DNA binding activity, as shown by DNase I footprint analysis, has only been observed by those who have reported no requirement for TFIIA [21,27,28]. These results are consistent with the suggestion that these investigators have copurified both TFIIA and TFIID activities. The physical properties of TFIIA have not been clearly elucidated. TFIIA activity was first purified as a single polypeptide of 43 kDa from HeLa cells [29] and was thought to be actin. Samuels and Sharp [30], from the isolation of TFIIA from calf thymus, suggested that

TABLE 1

Factor

Activities

Source of purification

Polypeptide composition

Purity

Equivalent

Ref.

llA

(1) Required for the formation

HeLa cell HeLa cell Calf thymus

9 43 kDa polypeptide 19.6: 19.1:12.8 kDa

2 200-fold ? 9CtYO-fold

STF AB

20 58 29

Drosophila

9

information not provided

Factor 1

79

HeLa cell HeLa cell

'~ '~

300-fold information

BTF1

20 58

HeLa cell

9

DB

19

,~e reference

liD

22

9

280- fold

! !D

!8

29.5 kDa

homogeneous

of a committed comples (2) Stabilizes l i D binding to TATA box

lID

(1) Binds to the TATA box

(2) Required for the formation of a committed complex (3) Positions RNA pol !!

subunils

not provided information

not provided yeast

23-27 kDa

polypeptide HeLa cell lib

IIF

(1) Required for preinitiation coinpl:x formation (heparin resistant complex) (2) Interacts with TFIIE/IIF

(1) Required for preinitiation complex formation. (heparin resistant complex) (2) Interacts ~ t h RNA pol I! (3) Affects RNA pol II elongation

HeLa cell

- "~

polypeptide Rat liver

35 kDa

homogeneous

a

46

BTF3

47

polypeptide HeLa eel ~.

27 kDa polypeptide

homogeneous

HeLa cell

78 and 30 kDa

2 200-fold

53

subunits HeLa cell

76 and 30 kDa

see reference

RAP30/76

40

homogeneous

Factor 5

38

homogeneous

BTF3

47

homogeneous

/2y

37

subunits

Drosophila

86 and 34 kDa

subunits HeLa cell

27 kDa

polypeptide Rat liver liE

(1) Required for preinitiation

HeLa cell

67 and 31 kDa subunits

13 O00-fi)ld

35

complex formation (heparin res;.s,.~nt complex) (2) Interacts with RNA pol II a Ha, I., Maldonado, E. and Reinberg. D.. unpublished data.

this activity eluted and sedimented as a 40 kDa species and determined that TFIIA activity was composed of three different polypeptides. Our studies with mammalian TFIIA indicated that the activity eluted from gel-filtration columns performed in high salt with apparent molecular masses of 84 and 44 kDa [20,48]. Roeder and co-workers [20] and Samuels and Sharp [30] isolated TFIIA activity devoid of actin. Also, purified preparations of actin did not contain TFIIA activity. Therefore, the suggestion that actin and TFIIA are the same proteins is unlikely. TFIIA activity has also been identified in Saccharomyces cerevisiae [49]; the molecular weight of this activity remains unknown. TFIIA was initially described as an activity required early in the transcription cycle [8,19,20]. Initially, Davidson et al. [8], indicated by DNA competition analysis using the conalbumin and the adenovirus major

late promoters that the formation of a DNA-TFilD complex was stabilized by TFIIA at either promoter [8]. Similar results were observed using the Ad-MLP and nonspecific competitors [19]. Also, it was demonstrated that the formation of the committed complex (defined by the formation of DNA protein complexes that undergo transcription in the presence of low concentrations of sarkosyl [31]) at the adenovirus major late promoter required TFIIA and TFIID [20,32]. Taken together, these observations strongly suggested that TFIIA stabilized TFIlD binding activity. A direct demonstration of this was accomplished by analyzing the binding activity of yeast TFilD to the adenovirus major late promoter. Buratowski and co-workers [49] demonstrated the formation of a stable TFllD-promoter complex that could be resolved by a gel mobility shift assay required TFiIA. Furthermore, each factor indepen-

dently failed to produce a stable DNA protein complex. The collective data strongly suggested that TFIIA is a bonafide transcription factor; however, the identity of the polypeptide(s) comprising TFIIA activity needs to be further investigated. IV. RNA polymerase I! binding proteins

kDa and 78 kDa polypeptides (Flores and Reinberg, unpublished data). An activity similar to TFIIF, based on polypeptide composition, has been isolated from rat liver [37], and Drosophila cells [38]. ~lso, Greenblatt and co-workers [39] have isolated by I~NA polymerase II affinity chromatogral~hy polypeptides of 30, 38 and 76 kDa, referred to as RAPs (RNA polymerase II

The binding of RNA polymerase II to the promoter requires a bound TFIID [19,20], and allows the entry of three other factors into the transcription cycle, TFIIB, TFIIE and TFIIF. The initial studies of Reinberg and Roeder [33], first demonstrated a direct interaction betg .en TFIIE aad RNA polymerase II. Subsequent studies determined that the TFIIE fraction used was composed of two transcription activities, TFIIE and TFIIF [34,35]. Both TFIIE and TFIIF independently interacted with purified RNA polymerase II [35]. Also, TFIIE and TFIIF activities formed a complex that could be resolved by glycerol gradient sedimentation (see Fig. 1). TFII E and TFIIF are required for the formation of rapid start complexes, defined by their ability to initiate transcription in the presence of heparin concentrations which are prohibitive of the activity of the free factor [35]. TFIIE activity eluted from high-salt gel-filtration columns and sedimented on glycerol gradients with an apparent molecular mass of 60-70 kDa. TFIIE has not yet been purified to homogeneity. On the other hand, TFIIF activity has been purified to apparent homogeneity and exists as a heterodimer of 30

50 A 40-

/

30"

o

2o o

E

@

W r,.0 Q. r~ 0

Q.

Fig. 1. Glycerol gradient analysis of the interaction between TFIIE, TFIIF and RNA polymerase II. RNA polymerase If, TFiIE and TFIIF were incubated and sedimented on a glycerol gradient (for experimental details, see Ref. 35). Panel A: aliquots of the gradient fractions were assayed for RNA polymerase II activity in a nonspecific transcription assay using single stranded DNA. Panel B: aliquots of the gradient fractions were assayed for both TFIIE and TFIIF activities in a specific transcription assay (TFIIE and TFIIF were omitte~ from the reconstituted assay). Panel C: aliquots of gradient fractions were assayed for TFIIE, TFIIF and RNA polymerase II complementing activity in a specific transcription assay (TFIIE, TFIIF and RNA polymerase II were omitted from the reaction). The products of the reaction were separated on a 4% polyacrylamide 7 M urea gel and the amounts of [a-32p]UMP incorporated into the specific transcripts were determined by excising and counting the bands from the gel. Panel D: autoradiograph of the gel containing the products of the assay shown in panel C. The sign + represents the complete reaction containing saturating amounts of RNA polymerase II, TFIIE, and TFIIF in addition to the other factors. Gradient fractions are numbered at the top of the panel. Panel E: aliquots of the gradient fractions were analyzed by Western blot using monoaffinity purified anti-RAP 30 antibodies. Control glycerol gradient experiments in which TFIIE and TFIIF were sedimented independently are not shown in this figure; however, we have previously demonstrated that both activities independently sedimented at the top of gradient under the conditions used in this experiment (see Ref. 35.).

A

PoIII

0.3 .B

. ltE.]rFI~

0.2 O.i

Q..

C

I

0.3

.

Pollr

0

IIE.nF.

0.2

0.I Q

-,.,.,.&,/ I0 20 30

40

D + I 4

6 8 I0 12 14 16 1 8 2 0 2 2 2 4 2 6 2 8

30 32 34 36 38 40

3 § 7 9 II 13 15 17 Ig 21 23 25 27 2931 33 35 38 3941

f

associating proteins). Furthermore, they observed an interaction of the 30 kDa protein (RAP 30) with the 76 kDa protein (RAP 76) using antibodies directed against RAP 30 [40]. It has also been demonstrated that both RAP 30 and RAP 76 were required to reconstitute ~ranscription [40]. Furthermore, antibodies directed against the RAP 30 protein cross-reacted with the 30 kDa polypeptide subunit of TFIIF [34]. Also, RAP 76 as well as the 78 kDa subunit of TFIIF were shown to be extensively phosphorylated [35,39,40]. These data strongly suggested that RAP 30 and RAP 76 constitute TFIIF activity. All the RNA polymerase II transcription systems thus far analyzed showed an absolute requirement for the hydrolysis for the ~-~, bond of ATP for productive initiation of transcription [41]. A marked inhibition of transcription was observed when fl-~,-imido-ATP (AMP-PNP) was substituted for ATP in the specific transcription reaction in vitro [41]. This ATP analog, wLfich can not be hydrolyzed at the fl-7 position, apparently failed to satisfy a requirement for hydrolyzable adenosine nucleotides during the initiation reaction. AMP-PNP can be hydrolyzed at the a-fl position, and post-initiation was incorporated normally into RNA during the elongation phase of the reaction. Addition of deoxyATP (dATP) to a transcription reaction conteining AMP-PNP restored the ability to initiate, even though the deoxy ATP can not be incorporated into the nascent RNA chain, clearly demonstrating that the requirement for the hydrolysis of the fl-y bond of ATP did not occur during elongation [42]. Previous studies have indicated that a crude fP, c::.o~ containing TFIIE and TFIIF also contained a DNA-dependent ATPase activity [26,42]. However, in our laboratory, we have not been able to detect ATPase activity in purified TFIIE and TFIIF fractions. Although it is clear that RNA synthesis in vitro requires the hydrolysis of the ~-'t bond of ATP, the component of the transcription macninery which mediates the ATP hydrolysis has not been identified. Reinberg and Roeder have demonstrated that the formation of a preinitiation complex did not require the hydrolysis of ATP [33]. This has been subsequently demonstrated by Conaway and Conaway [43] and by Arias and Dynan [44]. Also, the role of the hydrolysis of the fl-7 bond of ATP during transcription ir,,hiation has not been elucidated. The requirement for ATP hydrolysis during the initiation reaction may provide energy or serve as a phosphate donor in a phosphorylation reaction. TFIIB has been purified to near homogeneity and the activity has been identified as a single polypeptide of 27 kDa (Ref. 33 and Ha, Maldonado and Reinberg, unpublished data). A transcription factor of similar chromatographic properties and apparent molecular weight has been isolated from rat liver preparations [46]. Also, Zheng and co-workers [47], purified a factor

TFIOA DHA ,;'0

MLTF

TF|0A DNA TF||D ~....~po, ~ TFllA DNA TFIlD POL II TFilA DNA TFliD POL TFIIF TFII~: TFIIB

iNiTIATION

,,q;"" /

/

Fig. 2. Proposed model for the ordered assembly of transcription factors at the Ad-MLP. TFIIA represents the first factor that participates in the assembly of the preinitiation complex. Although kinetic analysis supports this concept, TFIIA has never been shown to contain any DNA binding activity (see Ref. 20). TFIID can then recognize the TATA box which results in the formation of the committed complex (TFIIA : DNA : TFIID). Subsequently, RNA polymerase !I followed by TFIIB, TFIIE and TFIIF associated with the c,omitted complex resulting in the formation of the preinitiation complex (TFIIA: DNA: T F i l D : POLl! : TFIIF: TFIIE: TFIIB). Initiation, as defined by the formation of the first phosphodiester bond, occurs in the presence of two ribonucleoside triphosphates. Subsequently, factors that participate in elongation enter into the transcription cycle (i.e., TFIIS, as described in Ref. 78). The circle represents recycling of RNA polyraerase II allowing multiple rounds of transcription. The bracketed area indicated the role of MLTF in stabilizing the committed complex.

of similar molecular weight, but that was functionally differenl from TFIIB. These investigators have demonstrated by glycerol gradient sedimentation that the purified polypeptide directly interacted with RNA polymerase If. On the other hand, our studies using antibody directed against TFIIB in combination with glycerol gradient sedimentation have failed to demonstrate a direct interaction of both activities. The observation of the inability of TFIIB to interact with RNA polymerase II is further supported by studies performed by Greenblatt and co-workers [40], using RNA polymerase II affinity chromatography. They demonstrated that extracts depleted of the RAP proteins could catalyze transcription upon the addition of the RAP 30 and RAP 76 polypeptides isolated from SDS-polyacrylamide gels. These results suggested that these extracts depleted of RAP contained TFIIB. The possibility remains that the 30 kDa polypeptide isolated by Zheng and co-workers [47] is the small subunit of TFIIF (RAP 30).

Analysis of transcription reactions using purified factors together with kinetic analyses has led to the formulation of a model by which the general transcription factors enter into the transcription cycle [20,48] (see Fig. 2). An alternative model was developed based on the isolation of different DNA protein complexes that could be resolved by DNA mobility shift assays by Buratowsky and co-workers [49]. The initial step, the recognition of the TATA motif by TFIID and TFIIA [20] (formation of a committed complex [31]), is commensurate with our model. However, these investigators postulated that the complex containing TFIID and TFIIA was recognized first by TFIIB, then by RNA polymerase II followed by TFIIE, -IIF. This conclusion was based on the fact that the addition of a partially purified TFIIB protein fraction to a DNA mobility shift assay containing TFIIA, -IID, and the adenoviras major late promoter (MLP) generated a more slowly migrating DNA-protein complex on nondenaturing polyacrylamide gels as compared to the TFIID, -IIA, MLP complex alone. On the other hand, our data have suggested that the committed complex is directly recognized by RNA polymerase II (see Fig. 2). In order to perform these experiments, Buratowsky and co-workers [49] used fractions containing TFIIB derived from fractionation of HeLa cell extracts on phosphocellulose followed by DEAE-Sephacel chroamtography. Our studies have demonstrated that these fractions, although enriched in TFIIB activity, also contained RAP 30 (TFIIF) reactive material (see Fig. 3 in Ref. 34). Also, Fire and co-workers [19] using partially purified fractions to generate transcription competent initiation complexes together with competition experiments, found that the committed complex was directly recognized by RNA polymerase II. The experiments of Buratowsky and co-workers [49] raise the possibility that TFIID forms a complex with TFIIB and that this allows the recognition of RNA polymerase II. Certainly, the elegant approach undertaken by Buratowski and co-workers combined with the use of purified factors would lead to a conclusive understanding of the factors constituting each step leading to the formation of the rapid start complex.

V. Transcription of non-TATA-sequence-containing promotel's

While it was originally thought that most of the RNA polymerase II transcribed genes contain the TATA motif, it has now become clear that there are a large number of class II genes lacking this recognition element. It is therefore of interest to determine how RNA polymerase II and associated factors operate in these promoters. We have extensively studied the adenovirus IVa2 promoter. The CAP site of this promoter is located approx. 210 nucleotides upstream from the CAP site of

the Ad-MLP. Transcription from the IVa2 and MLPs utilizes different DNA strands. Our initial studies focused on identifying a factor(s) that would allow the transcription of the I r a 2 promoter. Upon addition of saturating amounts of TFIIA, liB, liE, IIF and RNA polymerase II, in the absence of TFIID, we were unable to observe transcriptional activity. However, with the addition of limiting amounts of nuclear extract to the mixture containing the purified factors above, we were able to regain IVa2 specific transcription. Using this assay system, we attempted to purify the factor(s) responsible for IVa2 transcription. To our surprise we observed that the transcriptional stimulatory activity of the IVa2 promoter co-purified with TFIID activity [32]. Because the mammalian TFIID has not been purified to homogeneity, we were unable to unequivocally demonstrate that TFIID was required for the transcription of the TATA-Iess promoter. Therefore, ct~mpetition experiments were performed. These experiments showed that a promoter containing the TATA sequence (adenovirus EIV or major late promoters) effectively abolished transcription from the TATA-Iess promoter; however, the addition of a DNA containing a promoter with a deletion of the TATA motif failed to inhibit transcription. Inhibition of IVa2 transcription by plasmid DNA containing the TATA sequence could be overcome by the addition of excess amounts of protein fractions containing TFIID activity, the TATA binding protein [32]. These observations have led us to postulate that TFIID, the TATA binding protein, is required for transcription of the IVa2 promoter, a non-TATA-sequence-containing promoter [32]. We have further demonstrated that TFIID participates in the transcription of the IVa2 promoter by studying the yeast TFIID factor. Mammalian and yeast TFIID activities are known to have apparent molecular masses of 100 and 27 kDa, respectively [20,22,24]. Based on this molecular mass difference, we have been able to demonstrate that the mammalian activity capable of directing transcription from the IVa2 and major late promoters eluted from a high salt gel filtration column with an apparent molecular mass of approx. 100 kDa. On the other hand, when a similar experiment was performed using the yeast factor purified by gel-filtration chromatography, we were able to demonstrate that the transcriptional activities of the IVa2 and major late promoters eluted with an apparen', molecular mass of 27 kDa. In both cases the transcriptional activity cochromatographed with an activity capable of binding to the TATA sequence present in the Ad-MLP (Carcamo, J., Maldonado, E., Cortes, P., Ha, I. and Reinberg, D., unpublished data). Furthermore, we have been able to demonstrate that the yeast TFIID activity was capable of binding to sequences which overlapped with the CAP site of the IVa2 promoter (Carcamo et al., unpublished data). In addition we have also demonstrated that se-

quences overlapping the IVa2-CAP site can effectively compete for binding with yeast TFIID activity to the TATA sequence of the MLP (Carcamo et al., unpublished data). These experiments suggested that TFIID activity may recognize sequences other than the TATA motif. Experiments using alternative nonTATA sequence containing promoters may demonstrate that this phenomenon is of general importance. VL Specific transcription factors

It has been shown in vivo that transcription from different class II genes is regulated by upstream elements which serve as recognition sites for specific DNA binding proteins. Many different upstream elements as well as factors recognizing these elements have been identified [12,13,15,51]. The mode of interaction between the factors recognizing these elements with the basic transcription machinery remains an unresolved issue. Roeder and co-workers [52,53] have demonstrated that the binding of upstream factors - ATF (activating transcription factor) to the adenovirus EIV promoter, the yeast Gal 4 activating factor to a c.himeric construct containing Gal 4 recognition sites and the adenovirus EIV core promoter sequences - resulted in the modification of TFIID binding to the TATA sequence. These funda~nental experiments have led to the conceptualization that some upstream factors can operate through the modification of the general transcription factors. Our studies have focused on the mechanism by which factors recognizing upstream sequences activate transcription. Due to its simplicity, the adenovirus major late promoter, which contains only one upstream element [54], has provided a model framework by which we have functionally investigated the specific transcription factor referred to as major late transcription factor (MLTF) [55], (also know as USF [21], and UEF [56]). MLTF has been purified to apparent homogeneity by a number of groups [32,57-59]. The activity appears to be contained in a polypeptide of 46 kDa [32,57-59]. However, two independent reports have indicated the presence of two polypeptides of approx. 46 and 50 kDa present in highly purified preparations of MLTF [32,59]. Both polypeptides specifically bo,Jnd to the MLTF recognition site [32,59], and reacted with antibodies directed against a highly purified preparation of MLTF [59]. The relationship between these two polypeptides remains unknown. The initial observation of Sawadogo and Roeder [21] indicated that the addition of a partially purified MLTF preparation to a transcription system reconstituted with partially purified transcription factors and the adenovirus major late promoter resulted in a 10-fold stimulation of transcription. We have reproduced this observation using a highly purified preparation of

MLTF [32], and have observed that MLTF affected both the rate of the transcription reactions as well as the yield of RNA products. We have also demonstrated using the detergent sarkosyl as well as by competition experiments that MLTF directly affected the stability of the preinitiation complex [32]. Furthermore, it was demonstrated that MLTF stimulated only a 2-fold increase in transcription when the reactions were performed under conditions that prevented reinitiation [32]. Taken together, these studies suggested that MLTF was also capable of affecting a step subsequent to the formation of the pre-initiation complex. As indicated in the previous section, the binding of TFIID to the TATA motif serves as an entry site for RNA polymerase II into the transcription cycle. MLTF stabilizes the binding of TFIID and this permitted transcription reactions reconstituted with purified factors and the adenovirus major late promoter to undergo multiple rounds of transcription (Ref. 32, see Fig. 2). It was also of interest to examine the activity of MLTF at the nonTATA sequence containing promoter, Ad-IVa2. Because the CAP site of the IVa2 and major late promoters are separated by only 210 nucleotides and the transcription from each of these two promoters occurs on opposing DNA strands, we were able to examine whether the single upstream element, MLTF, located in between these two promoters could regulate the activity of both transcription units. Furthermore, Lennard and Egly [60] have demonstrated that the activity of the MLTF recognition site functioned in an orientation-independent manner to activate transcription at the major late promoter consistent with the dyad-symmetry sequence of the MLTF site. An 8-fold stimulation from the TATA-Iess promoter was observed [32]. The effect of MLTF on the simultaneous transcription of both promoters could not be studied because the sequences surrounding the CAP site of the major late promoter strongly inhibit the transcription from the IVa2 promoter [61]. The dyad symmetry of the MLTF site as well as the fact that this element can regulate transcription of both promoters strongly suggest that MLTF may bind as a dimer. However, it is surprising that experiments perfored by Lennard and Egly [60] and independently by Sawadogo and co-workers [59] and Hough and co-workers [62] using different methologies have resulted in the conclusion that only one protein molecule binds to this site. Unlike MLTF, the SP1 and CAAT box binding sites present in many genes, a second class of upstream element has been widely characterized that mediates the response to defined stimuli. One interesting example of a promoter that can mediate the response of some cellular genes to the second messenger cyclic AMP (cAMP) contains an upstream element that has been termed the cAMP response element (CRE) [63]. The CRE was first identified as the target sequence for a 43

kDa phosphoprotein isolated form PCI2 cells and termed CREB (cAMP responsive element binding protein) [64]. This transcription factor was shown to be regulated by phosphorylation via the cAMP-dependent protein kinase (PICA) as well as by protein kinase C (PKC) in vitro [65]. Since these initial findings, it has been shown that the CRE is also present in most of the adenovirus early genes (EIA, Eli, Elll, and EIV) whose expression is regulated by one of the products of the adenovirus early gene, EIA (p289) [15]. The CRE site can also mediate the response to EIA [66-69]. Interestingly, it was recently shown that the CRE serves as a rccognition site to multiple cellular proteins (Ref. 68 and Merino, A., Buckbinder, Lo, Mermelstein, F.H. and Reinberg, D., unpublished data). We have identified three families of factors which recognize and bind to the CRE. One factor that has been purified to homogeneity, EIVF (65-72 kDa), was shown to specifically bind to [he CRE present in the adenovirus early region IV promoter (Ref. 68 and Merino et al., unpublished data). This factor as well as one family with molecular masses ranging from 38 to 45 kDa and a third family in the 31-37 kDa range enhanced the basal level of transcription from the adenovims EIV promoter. We also observed that all three sets of factors were antigenically related to the transcription factor APl, the product of the cJUN oncogene [71-73]. APl recognizes a promoter element referred to as the TRE, named by virtue of the fact that it mediates the response of specific cellular genes to the phorbol ester tumor promoter, TPA [7134]. Karin [75] has discussed in a recent review the similarity between API and CREB, noting that the consensus sequence between the CRE (5'-TGACGTCA-3') and the API recognition sequence (5'-TGA[C or G]TCA-3') was remarkably similar, it was also observed that many of the proteins binding to both promoter elements fell within similar molecular mass ranges, for example 43 kDa for CREB and 44-47 kDa for API. On this basis, it has been suggested that extensive cross-talk occurs between these two signal transduction systems. Our data has demonstrated that phosphorylation regulates the binding affinity and specificity of the factors which recognize the CRE (Merino et al., unpublished data). For example, the DNA binding activity of EIVF was specifically regulated by phosphorylation with PICA in vitro (Merino et al., unpubfished data). This protein only recognized the CRE and failed to recognize the API site (Merino et al., unpublished data). In contrast, the binding specificity of proteins in the 45-38 and 31-37 kDa range was regulated by phosphogylation with PKA in vitro. Furthermore, we have demonstrated that the proteins in the 31-37 kDa family represent a single polypeptide that is differentially phosphorylated (Merino et al., unpublished data). The 31 kDa protein isolated from the cell bound only to the CRE; however, upon phosphorylation with PKA, the 31 kDa protein

was capable of recognizing both the CRE and AP1 sites. Two cDNA clones have been identified which represent two CRE binding proteins [76,77]. Interestingly, a comparison of the cDNA clones isolated revealed that there exists an internal deletion of 14 amino acids [76,77]. The protein sequences of both clones revealed that the proteins contained substrate domains for PKA, PKC and casein kinase !1, supporting the hypothesis that CREB can be regulated by phosphorylation and that multiple signal transduction pathways may converge on single substrates. Therefore, post-translational modification may, in part, regulate the interaction of specific factors with unique upstream elements. VII. Summary remarks Studies on the specific transcription of class il genes demonstrate that most of the enzymatic complexity as well as the regulation of RNA synthesis catalyzed by the RNA polymerase 11 transcription machinery probably resides in the initiation step. Many factors have been identified that regulate transcription, including those we have discussed above and many not included in this review. Protein fractions have been isolated that, when mixed together, have reconstituted specific transcription activity from many class !1 promoters. Characterization of the specific transcription factors has greatly been facilitated by the isolation of eDNA clones. The identification of the eDNA for the specific transcription [actors has successfully been accomplished through the use of oligonucleotides containing recognition sites for these factors [80]. The general transcription factors do not individually recognize specific sequences presen! in DNA and have been found in the cell m low abundance, making the isolation of eDNA clones slow and difficult. Only by purification of the general factors will it be possible to generate molecular probes (i.e., antibodies and/or oligonucleotides) required for the isolation of their respective genes. The identification of the genes coding for the general and specific transcription factors will readily lead to a greater understanding of the mode of action of these factors and the regulatory mechanisms underlying gene expression. Acknowledgements This work was supported by grants from: the National Institutes of Health Grant GM 37120, the National Science Foundation Grant DMB-8819342, the N.J. Commission on Cancer Research Grants 687-035 and 688-026 and a grant from the Foundation of the University of Meotcine and Dentistry of New Jersey. F.H.M. was supported by a taining grant from NIH No. ES07148. O.F. was supported by a fellowship from Schering Corp., D.R. was a recipient of an American Cancer Society J~mior Faculty Award (JFRA-205).

Re[erences ! Recder. R.G. (1976) in RNA P o l y m e r a ~ (Losick, R. and Chamberlin. M.. eds.), pp. 285-300, Cold Spring Harbor, NY, 2 Gi~inger. F.. Kedinger. C. and Chamh.~n, P. ~1974t Bi~chimie 56. 319-325. 3 Well, A.P., Luse, D,S., Segail, J. and Roeder, R.G. (1979) C¢11 18, 469-454. 4 Manley, J.L., Fire, A., Cano. A., Sharp, P.A. and Geher. M. (1980) Proc. Natl, Acad. Sci. U S A 77. 3855-3859, 5 Luse. D.S. and Roeder, R.G. (1980) Cell 20, 691-699. 6 Lee, D.C. and Rooder, R.G. (1981) Mol. Cell Biol. I, 635-651. 7 Matsui. T.. Segali. ,L, W¢il, A.P, and Roeder, R.G. (1980) J, Biol, Chem. 255, 11992-11996. 8 Davidson. B.L.. Egly, J.M., Mul,Ahill, E.R. and Chambon. P, (1983) Nature 301,680-686. 9 Samu¢ls, M., Fire, A. and Sharp. P.A. (1982) J. Biol. Chem, 257, 14419-14427. i0 Dynan. W.S. and Tijian, R. 0983) Cell 35, "~9-87. 11 Dignam, D.L., Levobi~,, R,M. and Roeder. R.G. (1983) Nucleic Acids Rcs. II. !475-1485. 12 Dynan. W.S. and Tijian, R. (1985) Nature 316. 774-778. 13 McKnight. S. and Tijian. R. (1987) Cell 46, 795~g05. 14 Breathnach. R, and Chambon, P. (1981) Annu. Rev. Bit~:hern. 50, 349-393. 15 Jones. N.C.. Rigby. P.W. and Ziff, F_B. {1988) Gene,,, [~v 2, 267-281. 16 KhouD', G., and Gruss. P. (1983) Cell 33. 313-314. 17 Sallzman. A,G. and Weinmann. g. (1989) F A S E B J. 3, 1723-1733, 18 Nakajima, N.. Horikoshi, M. and Roeder, R.G. (19881 $, Mot. Cell Biol, 8. 4028-40~). 19 Fire. A., Samuels, M. and Sharp, P,A, (198~i J. Biol ('hem+ 259. 2509-2516. 20 R¢inberg, D.. Horikoshi. M. and R ~ r , R.G. g1987) ,t. Biol. Chem. 262, 3322-3330. 21 Sawadogo, M. and Roeder. R.G. (1985) Cell 43. 165-175. 22 Buratowski, S., Hahn. S.. Sharp. P.A, and Guaren~e. L. ~19l~,t Nature 334. 37-42. 23 Cavallini. B,. Huet, $,. Plasmt, J.L.. Sentenac. A.. Egly. J.M. an~ Chambon, P. (1988) Nature 334. 77-80. 24 Samuds. M., F~rc, A.. and Sharp. P.A. (1982) ~. Biol. Chem, 257. 14419-14427. 26 Sawadogo. M. and Roeder. R.G. (1985) Proc. Natl. Acad. Sc~. USA 82. 4239-4398. 27 Van Dyke, M.W., Roeder, R.G. and Sawadogo. M. (1988) Science 241.1335- ! 338. 28 Hal, T., Horikoshi, M.. Roeder, R.G. and Green, M.R. (198~) Cell 54. 1043-1051. 29 Esly. J.M., Miyamoto, N.G.. Moncollin, V. and Chambon. P. (1984) EMBO J. 3. 2363-2371. 30 Samuels, M, and Sharp, P.A. (1986)J. Biol. Chem. 261, 2003-2013. 31 Hawley. D.K. and Roeder. R.G. (1985) J. Biol. Chem. 260, 8163-8172. 32 Carcamo, J.. Lobos, S., Merino. A.. Buckbinder, L., Weinmann. R., Venkatachala, N. and Reinberg, D. (1989) I. Biol. Chem. 264, 7"/04-7714. 33 Reinberg, D. and Roeder, R.G. (1987) J. Biol. Chem. 262. 3310-3321. 34 Flores, O., Maldonado, E., Burton, Z., Greenblatt. J. and Reinberg. D. (1988) J. Biol. Chem. 263, 10812. 35 Flores, O.. Maldonado. E, and Reinberg, D. (1989) ,L Biol. Chem. 264, 8913-8921. 37 Conaway. J.W. and Conaway. R.C, (1989) J. Biol. Chem. 264. 2357-2362.

38 Price, D.H., Sluder. A,E, and GreenJeaf. A.L. ~1989) Mol. Cell Biol. 9. 1465~ 1475. 39 Sopta, M,. Carthev,. R.W and Greenbla|l. J. (|985)J. Biol. Chem 26~, 10353-10360. 40 Burton. Z.E. Killeen. M.. ,~pta. M,. Or|o|an. L.G and Greenblau, J. (19881 Moi. Ceil. Biol. 8. i~)2-1613 41 Bunick, D., Zandomeni, r . Ackerman. S and Wemmann. R {1982) Cell 29, 877~8~6. 42 Sa~adogo, M and Roeder, R,G. (19841 J. Biol, Chem. 259. 5321-5326. ,*3 Conaway, R,C and Cona,,.'ay. J.W~ ~1987) J. Bml, Chem. 262, 2%2°2968. 44 Arias..LA. and Dynan, W,S. (1989)J. Biol, Chemo 2 ~ . 3223~3229. a~6 Conaway, JAW., [h~nd, M. and Conav,,ay. R (|987) ,1. Biol. ('hem. 262, 8293~8297. 47 gheng, X.M., Monco|in~ V., Eg~!,. J,M. and Chambon, P. (1987) Ceil 50. 361 ~368. ~. 48 Reinberg~ D,, Rotes. O, and Buckbmder, L ~1987) m Motecular Biology of RNA: Ne~' Pe~pcc|ivcs {|nouye, M, and Dud~:k. B,, ~Js,), pp. 423-439, Academic Pce~s. Nee,, York, ,~9 Buratov,ska. S.. Hahn. S.. Guaren|e. L and Sharp. PA. ~|9891 ('ell 56. 549~5bl. 51 Wingender. E. {i~88) Nucle0c Acids Res. 16, |879.~ 1 ~ i 52 Horikoshl, M., Carey, M.F., Kaksdam, t|, and R~der. R ( i {1988) O u 54. 6~5o669, 53 Honkoshi, M.. HaL T.. Lin. Y.S.. Green. MR. and goeder. R.G (1988) ('ell $4. 1033-1042. 54 Hu. S. and Manley. J.L (19811 Pr~. Nail. Acid. S~a. USA 78. 820-824. 55 Carth¢~'. g.W.. Chodosh. L.A. and Sharp. PA. (19851 Cell 43, 439-446. 56 Mi~,a~m~lo, N.G., Mon~:~lin. V., Wm~en~h. M. Hen, R. Egb, J,M, and Chamtmn, P. (198~) Nuc!e~c Ac~d~ Rcs. 12. 8779~8799 57 Ch~dosh. L.A.. Ca~h~',v. R,W. and Sharp, P.A, ~198f~) Mol. Cell. Biol. 6. 4723~4733, 58 Moncx~llin. V.. Miyamoto, N.G. Zhcng. X,M. ~nd |'~gb. J M. {1986) EMBO. ~. 5. 2577-25M. 59 Sav.,adogo, M.. Van Dyke. M.W.. Gregor. P.D. and R~der. R.G 1988) ~. Biol. ('hem. 263. 11985-11993. t~ Lennard. A.C and Egly. J,M. (19871 EMBO J 6, ,~)27~~ 61 Natarajan. V., Maden, M.J. and Saizman. N.P. (19851J. V~rol. 55. IO-15, 62 Hough, P.V.C. Mastrang¢lo, I.A. Wall, J.S.. Haimfield, ~,F.. Sawadogo. M. and Roeder, R.G. (1987) Pro¢. Natl. Acid Set, USA 84 4826-~830. 63 Montmmy, M.R,. Scvanno. K.A., Wagner, J.A., Mand¢|. G. and Goodman, H.M. (1986) Pica:. Natl. Acid. Sci. USA 83. 6682-6~86. 64 Monlminy. M.R, and Bilc~kjian, L.M. (1987)Nature 328,1"~5-178. 65 Yamamoto, K.K., Gonzales, G.A., Biggs. W.H.. ill and Montminy, M.R. (1988l Nature 334, 494-498. 66 Lee, K.A.W. and Green. M.R. (1987) EMBO $, 6, 1~5-1333. 67 Leza. A. and Heating, P. (1988) J. Virology 62. 3003+3013. 68 Cortes. P., Buckbinder, L., [x~_a, A,, Rak, N., Hearing. P.. Menno. A. and Reinherg, D. 0988) Genes Dcv. 2, 975-990. 69 Miralles, V.,L. Cortes, PC.. Stone. N. and Reinbcrg. D. (19891 ~. Biol. Chem. 264. 10763-10772. 71 Angel. P.. imagawa. M.. Chiu. R.. Stem, B.. imbra, gJ., gahmsdoff. HJ., Jonat, C., HerHich, P. and Karin, M. (1987) Cell ~9. 729-739. 72 Vogt. P.K. and Tijan, R. (1988) Oncogene 3, 3-7. 73 Vogt. P.K. and Bos, T.J. (1989) Trends Biochem. Sci. 14. 172-175. 74 Chiu. R.. lmagawa, M.. lmbra. R.G.. Bockoren. J.R. and Kann. M. (1987) Nature 329, 648-651. 75 Karin, M. (1989) Trends Genetics 5, 65-67. 76 Hoeffler. J.P.. Kotwelman. R. and Rocder, R.G. (1988) Science 242, 1430-1433.

10 77 Gonzalez, G.A., Yamamoto, K.K., Fischer, W.H., Karr, D., Menzel, P., Biggs, W., Vale, W.W. and Montiminy, M.R. (1989) Nature 337, 749-752. 78 Reinberg, D. and Roeder, R.G. (1987) J. Biol. Chem. 262, 3331-3337.

79 Price, D.H., Sluder, A.E. and Greenleaf, A.L. (1987) J. Biol. Chem. 262, 3244-3255. 80 Singh, H., LeBowitz, J.H., Baldwin, A.S. and Sharp, P.A. (1988) Cell 52, 415-423.