Cell, Vol. 66, 1067-1070,
September
20, 1991, Copyright
0 1991 by Cell Press
Roles of TFIID in Transcriptional Initiation by RNA Polymerase II Jack Greenblatt Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics University of Toronto Toronto, Ontario Canada M5G IL6
Transcription factor IID (TFIID) is a site-specific, DNAbinding complex required for selective initiation of transcription by eukaryotic RNA polymerase II. Promoters for RNA polymerase II often contain a TATA box, recognized by TFIID, located a short distance (25-30 bp in mammals) upstream of the transcriptional initiation site. A TFIID-promoter complex directs the ordered assembly onto the promoter of RNA polymerase II and other general initiation factors (TFIIA or TFIIG, TFIIB, TFIIE, and TFIIF) to create a multiprotein complex capable of transcriptional initiation (reviewed by Sawadogo and Sentenac, 1990). This ordered assembly pathway, or a component(s) in the fully assembled initiation complex, is the ultimate target of regulatory processes leading to altered transcription of specific genes. Recent evidence suggests that one important target for the activation domains of particular regulatory proteins in the proximal initiation complex is TFIID. TFIID: Structure and DNA Binding The molecular weight of partially purified human or Drosophila TFIID is much larger than the molecular weight of the human or Drosophila polypeptide that binds to the TATA box, which I shall refer to as TATA-binding protein (TBP) (Hoffmann et al., 1990; Hoeyet al., 1990; Kao et al., 1990; Peterson et al., 1990; Dynlacht et al., 1991). Natural TFIID appears to be a multisubunit protein: Drosophila TBP is tightly associated with at least six other polypeptides (Dynlacht et al., 1991). Complementary DNAs encoding TBP have been cloned from yeast, insects, plants, and mammals, and a schematic diagram of TBP’s structure is shown in Figure la. For a TATA box-containing promoter, the C-terminal 180 amino acids of TBP can replace a TFIID fraction for basal transcription in vitro (Hoey et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). This part of TBP is at least 80% identical in all organisms (see, for example, Peterson et al., 1990; Gasch et al., 1990) and behaves like a single domain that is completely inactivated for DNA-binding and basal transcription activities by any small deletion (Horikoshi et al., 1990). Indeed, yeast and human TBP are functionally interchangeable for basal transcription in yeast and human systems in vitro (reviewed by Sawadogo and Sentenac, 1990). Nevertheless, yeast cannot survive with the C-terminal region of human TBP (Cormack et al., 1991; Gill and Tjian, 1991), although the significance of this species incompatibility is still obscure. The N-terminal regions of TBPs from different species vary greatly in size (e.g., 18 amino acids in one form of TBP from the plant Arabidopsis thaliana, 159 amino acids in humans), sequence, and amino acid composition. The
Minireview
N-terminal region has no essential role in transcription, at least in yeast: the budding yeast Saccharomyces cerevisiae grows well when its TBP gene is replaced with the TBP gene from the fission yeast Schizosaccharomyces pombe, which has a very different N-terminal region (Fikes et al., 1990). In addition, at least some strains of S. cerevisiae can grow when the N-terminal region of TBP is either completely missing (Cormack et al., 1991; Gill and Tjian, 1991; Poon et al., 1991; Reddy and Hahn, 1991; Zhou et al., 1991) or replaced with a very different N-terminal region of human origin (Cormack et al., 1991; Gill and Tjian, 1991). The C-terminal region of TBP contains two DNA-binding 66-67 amino acid repeats (amino acids 67-l 31 and 157222 in yeast TBP) that are nearly 40% identical and flank a basic region (Figure 1 a). Since TBP binds to DNA as a monomer (Horikoshi et al., 1990) each of the two repeat motifs in a single TBP molecule likely binds to a part of the TATA box. As pointed out by Reddy and Hahn (1991) the repeats in TBP could recognize the TATA box either as an imperfect direct repeat or as an imperfect inverted repeat (Figures 1 b and lc). In either case, the inherently asymmetrical nature of most TATA box sequences (e.g., TATAAA) together with the likely nonequivalence of the imperfect repeats in TBP should enable the orientation of DNA-bound TBP to define the direction of transcription. TFIID binds TATA sequence-containing DNA about 105-fold more strongly than random sequence DNA, and variant forms of the TATA box are bound by yeast TFIID with similar dissociation constants (2-4 x 1 Oe9M)(Hahn et al., 1989). With the exception of Arabidopsis, in which two forms of TBP seem to recognize identical sequences (Gasch et al., 1990), most eukaryotes seem to produce only one form of TBP. This must therefore recognize the
(b)
(4
Figure 1. Structure
and DNA Binding of TBP
(a) Structural features of TBP. (b) Recognition of the TATA box as an imperfect inverted repeat. (c) Recognition of the TATA box as an imperfect direct repeat.
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Figure 2. Assembly a Promoter
TFIIB
of TBP and Other General Initiation Factors onto
variant TATA sequences, and there is no evidence for the existence of multiple forms of TBP that might recognize different TATA sequences. TFllD as a Genera/ initiation Factor Under some conditions the TBP-promoter interaction is stable only in the presence of a second general initiation factor, either TFIIA or TFIIB (Buratowski et al., 1989). These are partially purified fractions, and therefore the issue of what proteins are interacting directly or indirectly with TBP remains unclear. The association of TFIIB with a TBP-promoter complex, in the presence or absence of TFIIA, creates a preinitiation complex that can recruit RNA polymerase II, either alone (Buratowski et al., 1989) or in association with the small subunit RAP30 of the RNA polymerase II-binding general initiation factor RAP30/74, also known as TFIIF (Buratowski et al., 1991; Flores et al., 1991). Therefore, RNA polymerase II and/or RAP30 should interact with TFIIB or TBP (Figure 2). Work in progress may soon define direct interactions of TBP with other general initiation factors. Is TBP required to provide essential protein-protein interactions for initiation by RNA polymerase II when the promoter has an initiator element around the transcriptional initiation site but no upstream TATA box? A TFIID fraction is needed for initiation in vitro at such promoters @male et al., 1990, and references therein), but the involvement of TBP and its associated polypeptides will not be unambiguous until it is possible to replace them with bacterially produced recombinant proteins. Part of the TBP amino acid sequence (residues 197-240 in S. cerevisiae TBP) overlapping its second DNA-binding repeat is somewhat similar to aconserved region in bacterial o factors (Horikoshi et al., 1989) (Figure la). The best match involves part of TBP in which three point mutations affect DNA binding (Reddy and Hahn, 1991) and part of o that recognizes the -10 region in eubacterial promoters (TATAAT for a”). Thus TBP may have preserved a part of o responsible for promoter recognition, while other functional domains of o could be distributed among other general initiation factors (e.g., RAP30) that associatewith RNA
polymerase II via protein-protein interactions (McCracken and Greenblatt, 1991). TFllD and Transcriptional Regulation Many activator proteins can be dissected into two functional domains: one binds to DNA, and the other somehow activates transcription of the linked gene (reviewed by Ptashne, 1988). There are at least several chemically distinct kinds of activation domains, including some that are highly acidic (e.g., GAL4 and GCN4 of yeast and VP16 of herpes simplex virus), some that are glutamine-rich (e.g., Spl), and some that are proline-rich (e.g., CTF) (reviewed by Mitchell and Tjian, 1989). The ability of acidic activation domains to function in a variety of eukaryotes implies that their mechanism of activation is universal. The ability of particular activation domains to inhibit (“squelch”) the activities of some, but not all, other kinds of activation domains in cotransfection experiments suggests there could be multiple pathways leading to transcriptional activation (Tasset et al., 1990; Martin et al., 1990). That would be not unlike the situation in bacteria, where some activators aid binding of RNA polymerase to the promoter, and others aid melting of the promoter DNA by the RNA polymerase. One early indication that TFIID might be involved in transcription activation was the observation that activation by the adenovirus ElA protein at certain promoters depended on the sequence of the TATA box (Wu et al., 1987; Simon et al., 1988). Since there seems to be only one form of mammalian TBP (see Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990) there could exist multiple, TATA sequence-dependent conformations of TFIID that interact differently with certain activator proteins (or their downstream effecters). In fact, activation by ElA seems to result from direct interaction of its activation domain with the conserved region of human TBP (Horikoshi et al., 1991; Lee et al., 1991). Other recent experiments suggest that interactions between certain activator proteins and TFIID could prevent repression of transcription in vitro by histone Hl or assembled nucleosomes (Figure 3a). Assembled nucleosomes appear to repress transcription in vivo in yeast (Han and Grunstein, 1988) and also block accurate initiation, but not chain elongation, by RNA polymerase II in cell-free systems. When partially purified human TFIID, or even bacterially produced yeast TBP, is prebound to a promoter, it can prevent repression caused by nucleosome assembly in vitro (Meisterernst et al., 1990, and references therein). This ability of TFIID to prevent repression is enhanced by at least several activator proteins: the pseudorabies virus IE protein, USF (or MLTF), Spl, the GAGA factor, and GAL4-VP16 (a chimeric protein with the DNAbinding domain of GAL4 and the highly acidic activation domain of VP16) (Workman et al., 1991; Croston et al., 1991, and references therein). Alleviation of repression by pseudorabies IE protein requires only the TATA box region of the adenovirus major late promoter, while the other regulators require their specific binding sites in order to function. USF, a factor that binds to the adenovirus major late promoter upstream of the TATA box, can cooperate with both partially purified human TFIID and recombinant yeast
antiremession
TFiiA
w
Figure 3. Models for the Involvement vation
of TFIID in Transcriptional
Acti-
(a) Activator protein interactions with TFIID may prevent repression by histones. (b) Activator protein interactions with both TFIIB and TBP could lead to synergistic activation of transcription. (c) Possible roles for TBP-associated factors or other mediator proteins as adaptors or bridges.
TBP to prevent repression during nucleosome assembly. Partially purified human TFllD also stabilizes the binding of USF to the adenovirus major late promoter. This suggests that USF may directly interact with TBP in order to relieve repression by chromatin (Meisterernst et al., 1990; Workman et al., 1990, and references therein). GAL4-VP16 can prevent repression by both nucleoSomers (Workman et al., 1991) and histone Hl (Croston et al., 1991) and the acidic activation domain of VP16 is
necessary for antirepression by GAL4-VP16 (Workman et al., 1991). Stimulation of transcription by GAL4-VP16 in nucleosome assembly conditions occurs with both human TFIID and recombinant yeast TBP (Workman et al., 1991). Moreover, the activation domain of VP1 6 can bind directly to yeast TBP, and this VP1 6-TBP interaction seems biologically meaningful, since inactivating mutations in VP16 that do not change its charge prevent binding of VP16 to TBP (Ingles et al., 1991). The ability of VP16 to function with both human and yeast TFIID suggests that the VP1 6binding site is in the conserved C-terminal portion of TBP, and is consistent with the ability of acidic activators to function in yeast without the nonconserved, N-terminal region of TBP (Cormack et al., 1991; Gill and Tjian, 1991; Poon et al., 1991; Reddy and Hahn, 1991). Thus the antirepressing effects of GAL4-VP16 and many other activator proteins in chromatin assembly systems are most simply explained by postulating that their activation domains interact with TFIID to preserve or create a TFIID-containing, nonrepressible preinitiation complex that can promote multiple rounds of initiation by RNA polymerase II. The model shown in Figure 3a defines a single target for transcriptional activation domains, namely, TFIID. Thus, it cannot explain why activation by multiple, DNA-bound activation domains, including that of VP1 6, is usually synergistic (reviewed by Ptashne, 1988; also see Carey et al., 1990; Lin et al., 1990, and references therein). The model in Figure 3a also involves repression by histones and does not explain how activation can occur in cell-free systems in the apparent absence of chromosomal proteins. Thus, a recent observation that the activation domain of VP16 can bind a second general initiation factor, TFIIB, is very striking (Lin and Green, 1991). In that work also, a partially purified TFIIB fraction was used, and so the precise interactions of VP16 with a component(s) of the fraction are unclear. Nevertheless, the ability of VP1 6 to interact with both TBP (Stringer et al., 1990) and a second general initiation factor (Lin and Green, 1991) leads naturally to a model that explains synergistic activation of transcription (Figure 3b). Two DNA-bound activation domains could accelerate an otherwise slow step in the assembly of the RNA polymerase II initiation complex by bringing together TBP and TFIIB. Assembly of RNA polymerase II and the remaining general intiation factors into the initiation complex would then be possible (Buratowski et al., 1989; Lin and Green, 1991). This direct activation of the general initiation pathway by GAL4-VP16 would be superimposed on its ability to prevent repression by nucleosomes, generating the very substantial activation of transcription by GAL4-VP16 that is observed when histone Hl is present or nucleosome assembly is coupled to transcription (Meisterernst et al., 1990; Workman et al., 1991; Croston et al., 1991, and references therein). Recently, it was suggested that several activator proteins, including Spl , USF, and GAL4-VP16, require a “coactivator” or “mediator” in order to activate transcription in vitro. In the case of VP16, the mediator, which apparently can bind toVP16(Kelleheret al., 1990; Bergeret al., 1990) could most simply be envisioned as an adaptor that couples VP16 to a general initiation factor like TFIIB, as a
Cell 1070
subunit of TFIID other than TBP, or as a bridge that holds two VP16 activation domains the correct distance apart to bind TBP and TFIIB simultaneously (Figure 3~). Alternatively, the mediator could be a novel VPlG-binding form of one of the general initiation factors. Is the model shown in Figure 3b appropriate for activators other than GAL4-VP16? Like GAL4-VP16, USF likely achieves antirepression by interacting with TFIID (Meisterernst et al., 1990). In addition, it requires at least one additional factor, which strongly inhibits basal transcription and may interact with human TBP, in order to activate transcription in the absence of nucleosomes (Hoffmann et al., 1990; Meisterernst et al., 1990, 1991). Similarly, the Drosophila activator NTF-1 requires at least one factor tightly bound to TBP, possibly a subunit of native TFIID, in order to activate transcription (Dynlacht et al., 1991). In the case of Spl , its antirepressing effect (Croston et al., 1991) could well reflect an interaction with TFIID. In addition, the N-terminal, nonconserved portion of TBP and at least one factor that associates with TBP seem to be important for activation by Spl in nonchromatin systems (Dynlacht et al., 1991; Meisterernst et al., 1991, and references therein). In at least some of these cases, the activator protein may activate transcription by relieving inhibition caused by factors that associate with TBP (Meisterernst et al., 1991). More generally, recent and continuing progress in the cloning of cDNAs encoding all the general initiation factors should lead naturally to more precise models for transcriptional activation. References Berger, S. L., Cress, W. D., Cress, A., Triezenberg, ente, L. (1990). Cell 61, 1199-1208. Buratowski, S., Hahn, S., Guarente, 56, 549-561.
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September
20, 1991, Copyright
0 1991 by Cell Press
Roles of TFIID in Transcriptional Initiation by RNA Polymerase II Jack Greenblatt Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics University of Toronto Toronto, Ontario Canada M5G IL6
Transcription factor IID (TFIID) is a site-specific, DNAbinding complex required for selective initiation of transcription by eukaryotic RNA polymerase II. Promoters for RNA polymerase II often contain a TATA box, recognized by TFIID, located a short distance (25-30 bp in mammals) upstream of the transcriptional initiation site. A TFIID-promoter complex directs the ordered assembly onto the promoter of RNA polymerase II and other general initiation factors (TFIIA or TFIIG, TFIIB, TFIIE, and TFIIF) to create a multiprotein complex capable of transcriptional initiation (reviewed by Sawadogo and Sentenac, 1990). This ordered assembly pathway, or a component(s) in the fully assembled initiation complex, is the ultimate target of regulatory processes leading to altered transcription of specific genes. Recent evidence suggests that one important target for the activation domains of particular regulatory proteins in the proximal initiation complex is TFIID. TFIID: Structure and DNA Binding The molecular weight of partially purified human or Drosophila TFIID is much larger than the molecular weight of the human or Drosophila polypeptide that binds to the TATA box, which I shall refer to as TATA-binding protein (TBP) (Hoffmann et al., 1990; Hoeyet al., 1990; Kao et al., 1990; Peterson et al., 1990; Dynlacht et al., 1991). Natural TFIID appears to be a multisubunit protein: Drosophila TBP is tightly associated with at least six other polypeptides (Dynlacht et al., 1991). Complementary DNAs encoding TBP have been cloned from yeast, insects, plants, and mammals, and a schematic diagram of TBP’s structure is shown in Figure la. For a TATA box-containing promoter, the C-terminal 180 amino acids of TBP can replace a TFIID fraction for basal transcription in vitro (Hoey et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). This part of TBP is at least 80% identical in all organisms (see, for example, Peterson et al., 1990; Gasch et al., 1990) and behaves like a single domain that is completely inactivated for DNA-binding and basal transcription activities by any small deletion (Horikoshi et al., 1990). Indeed, yeast and human TBP are functionally interchangeable for basal transcription in yeast and human systems in vitro (reviewed by Sawadogo and Sentenac, 1990). Nevertheless, yeast cannot survive with the C-terminal region of human TBP (Cormack et al., 1991; Gill and Tjian, 1991), although the significance of this species incompatibility is still obscure. The N-terminal regions of TBPs from different species vary greatly in size (e.g., 18 amino acids in one form of TBP from the plant Arabidopsis thaliana, 159 amino acids in humans), sequence, and amino acid composition. The
Minireview
N-terminal region has no essential role in transcription, at least in yeast: the budding yeast Saccharomyces cerevisiae grows well when its TBP gene is replaced with the TBP gene from the fission yeast Schizosaccharomyces pombe, which has a very different N-terminal region (Fikes et al., 1990). In addition, at least some strains of S. cerevisiae can grow when the N-terminal region of TBP is either completely missing (Cormack et al., 1991; Gill and Tjian, 1991; Poon et al., 1991; Reddy and Hahn, 1991; Zhou et al., 1991) or replaced with a very different N-terminal region of human origin (Cormack et al., 1991; Gill and Tjian, 1991). The C-terminal region of TBP contains two DNA-binding 66-67 amino acid repeats (amino acids 67-l 31 and 157222 in yeast TBP) that are nearly 40% identical and flank a basic region (Figure 1 a). Since TBP binds to DNA as a monomer (Horikoshi et al., 1990) each of the two repeat motifs in a single TBP molecule likely binds to a part of the TATA box. As pointed out by Reddy and Hahn (1991) the repeats in TBP could recognize the TATA box either as an imperfect direct repeat or as an imperfect inverted repeat (Figures 1 b and lc). In either case, the inherently asymmetrical nature of most TATA box sequences (e.g., TATAAA) together with the likely nonequivalence of the imperfect repeats in TBP should enable the orientation of DNA-bound TBP to define the direction of transcription. TFIID binds TATA sequence-containing DNA about 105-fold more strongly than random sequence DNA, and variant forms of the TATA box are bound by yeast TFIID with similar dissociation constants (2-4 x 1 Oe9M)(Hahn et al., 1989). With the exception of Arabidopsis, in which two forms of TBP seem to recognize identical sequences (Gasch et al., 1990), most eukaryotes seem to produce only one form of TBP. This must therefore recognize the
(b)
(4
Figure 1. Structure
and DNA Binding of TBP
(a) Structural features of TBP. (b) Recognition of the TATA box as an imperfect inverted repeat. (c) Recognition of the TATA box as an imperfect direct repeat.
Cell 1066
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Figure 2. Assembly a Promoter
TFIIB
of TBP and Other General Initiation Factors onto
variant TATA sequences, and there is no evidence for the existence of multiple forms of TBP that might recognize different TATA sequences. TFllD as a Genera/ initiation Factor Under some conditions the TBP-promoter interaction is stable only in the presence of a second general initiation factor, either TFIIA or TFIIB (Buratowski et al., 1989). These are partially purified fractions, and therefore the issue of what proteins are interacting directly or indirectly with TBP remains unclear. The association of TFIIB with a TBP-promoter complex, in the presence or absence of TFIIA, creates a preinitiation complex that can recruit RNA polymerase II, either alone (Buratowski et al., 1989) or in association with the small subunit RAP30 of the RNA polymerase II-binding general initiation factor RAP30/74, also known as TFIIF (Buratowski et al., 1991; Flores et al., 1991). Therefore, RNA polymerase II and/or RAP30 should interact with TFIIB or TBP (Figure 2). Work in progress may soon define direct interactions of TBP with other general initiation factors. Is TBP required to provide essential protein-protein interactions for initiation by RNA polymerase II when the promoter has an initiator element around the transcriptional initiation site but no upstream TATA box? A TFIID fraction is needed for initiation in vitro at such promoters @male et al., 1990, and references therein), but the involvement of TBP and its associated polypeptides will not be unambiguous until it is possible to replace them with bacterially produced recombinant proteins. Part of the TBP amino acid sequence (residues 197-240 in S. cerevisiae TBP) overlapping its second DNA-binding repeat is somewhat similar to aconserved region in bacterial o factors (Horikoshi et al., 1989) (Figure la). The best match involves part of TBP in which three point mutations affect DNA binding (Reddy and Hahn, 1991) and part of o that recognizes the -10 region in eubacterial promoters (TATAAT for a”). Thus TBP may have preserved a part of o responsible for promoter recognition, while other functional domains of o could be distributed among other general initiation factors (e.g., RAP30) that associatewith RNA
polymerase II via protein-protein interactions (McCracken and Greenblatt, 1991). TFllD and Transcriptional Regulation Many activator proteins can be dissected into two functional domains: one binds to DNA, and the other somehow activates transcription of the linked gene (reviewed by Ptashne, 1988). There are at least several chemically distinct kinds of activation domains, including some that are highly acidic (e.g., GAL4 and GCN4 of yeast and VP16 of herpes simplex virus), some that are glutamine-rich (e.g., Spl), and some that are proline-rich (e.g., CTF) (reviewed by Mitchell and Tjian, 1989). The ability of acidic activation domains to function in a variety of eukaryotes implies that their mechanism of activation is universal. The ability of particular activation domains to inhibit (“squelch”) the activities of some, but not all, other kinds of activation domains in cotransfection experiments suggests there could be multiple pathways leading to transcriptional activation (Tasset et al., 1990; Martin et al., 1990). That would be not unlike the situation in bacteria, where some activators aid binding of RNA polymerase to the promoter, and others aid melting of the promoter DNA by the RNA polymerase. One early indication that TFIID might be involved in transcription activation was the observation that activation by the adenovirus ElA protein at certain promoters depended on the sequence of the TATA box (Wu et al., 1987; Simon et al., 1988). Since there seems to be only one form of mammalian TBP (see Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990) there could exist multiple, TATA sequence-dependent conformations of TFIID that interact differently with certain activator proteins (or their downstream effecters). In fact, activation by ElA seems to result from direct interaction of its activation domain with the conserved region of human TBP (Horikoshi et al., 1991; Lee et al., 1991). Other recent experiments suggest that interactions between certain activator proteins and TFIID could prevent repression of transcription in vitro by histone Hl or assembled nucleosomes (Figure 3a). Assembled nucleosomes appear to repress transcription in vivo in yeast (Han and Grunstein, 1988) and also block accurate initiation, but not chain elongation, by RNA polymerase II in cell-free systems. When partially purified human TFIID, or even bacterially produced yeast TBP, is prebound to a promoter, it can prevent repression caused by nucleosome assembly in vitro (Meisterernst et al., 1990, and references therein). This ability of TFIID to prevent repression is enhanced by at least several activator proteins: the pseudorabies virus IE protein, USF (or MLTF), Spl, the GAGA factor, and GAL4-VP16 (a chimeric protein with the DNAbinding domain of GAL4 and the highly acidic activation domain of VP16) (Workman et al., 1991; Croston et al., 1991, and references therein). Alleviation of repression by pseudorabies IE protein requires only the TATA box region of the adenovirus major late promoter, while the other regulators require their specific binding sites in order to function. USF, a factor that binds to the adenovirus major late promoter upstream of the TATA box, can cooperate with both partially purified human TFIID and recombinant yeast
antiremession
TFiiA
w
Figure 3. Models for the Involvement vation
of TFIID in Transcriptional
Acti-
(a) Activator protein interactions with TFIID may prevent repression by histones. (b) Activator protein interactions with both TFIIB and TBP could lead to synergistic activation of transcription. (c) Possible roles for TBP-associated factors or other mediator proteins as adaptors or bridges.
TBP to prevent repression during nucleosome assembly. Partially purified human TFllD also stabilizes the binding of USF to the adenovirus major late promoter. This suggests that USF may directly interact with TBP in order to relieve repression by chromatin (Meisterernst et al., 1990; Workman et al., 1990, and references therein). GAL4-VP16 can prevent repression by both nucleoSomers (Workman et al., 1991) and histone Hl (Croston et al., 1991) and the acidic activation domain of VP16 is
necessary for antirepression by GAL4-VP16 (Workman et al., 1991). Stimulation of transcription by GAL4-VP16 in nucleosome assembly conditions occurs with both human TFIID and recombinant yeast TBP (Workman et al., 1991). Moreover, the activation domain of VP1 6 can bind directly to yeast TBP, and this VP1 6-TBP interaction seems biologically meaningful, since inactivating mutations in VP16 that do not change its charge prevent binding of VP16 to TBP (Ingles et al., 1991). The ability of VP16 to function with both human and yeast TFIID suggests that the VP1 6binding site is in the conserved C-terminal portion of TBP, and is consistent with the ability of acidic activators to function in yeast without the nonconserved, N-terminal region of TBP (Cormack et al., 1991; Gill and Tjian, 1991; Poon et al., 1991; Reddy and Hahn, 1991). Thus the antirepressing effects of GAL4-VP16 and many other activator proteins in chromatin assembly systems are most simply explained by postulating that their activation domains interact with TFIID to preserve or create a TFIID-containing, nonrepressible preinitiation complex that can promote multiple rounds of initiation by RNA polymerase II. The model shown in Figure 3a defines a single target for transcriptional activation domains, namely, TFIID. Thus, it cannot explain why activation by multiple, DNA-bound activation domains, including that of VP1 6, is usually synergistic (reviewed by Ptashne, 1988; also see Carey et al., 1990; Lin et al., 1990, and references therein). The model in Figure 3a also involves repression by histones and does not explain how activation can occur in cell-free systems in the apparent absence of chromosomal proteins. Thus, a recent observation that the activation domain of VP16 can bind a second general initiation factor, TFIIB, is very striking (Lin and Green, 1991). In that work also, a partially purified TFIIB fraction was used, and so the precise interactions of VP16 with a component(s) of the fraction are unclear. Nevertheless, the ability of VP1 6 to interact with both TBP (Stringer et al., 1990) and a second general initiation factor (Lin and Green, 1991) leads naturally to a model that explains synergistic activation of transcription (Figure 3b). Two DNA-bound activation domains could accelerate an otherwise slow step in the assembly of the RNA polymerase II initiation complex by bringing together TBP and TFIIB. Assembly of RNA polymerase II and the remaining general intiation factors into the initiation complex would then be possible (Buratowski et al., 1989; Lin and Green, 1991). This direct activation of the general initiation pathway by GAL4-VP16 would be superimposed on its ability to prevent repression by nucleosomes, generating the very substantial activation of transcription by GAL4-VP16 that is observed when histone Hl is present or nucleosome assembly is coupled to transcription (Meisterernst et al., 1990; Workman et al., 1991; Croston et al., 1991, and references therein). Recently, it was suggested that several activator proteins, including Spl , USF, and GAL4-VP16, require a “coactivator” or “mediator” in order to activate transcription in vitro. In the case of VP16, the mediator, which apparently can bind toVP16(Kelleheret al., 1990; Bergeret al., 1990) could most simply be envisioned as an adaptor that couples VP16 to a general initiation factor like TFIIB, as a
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subunit of TFIID other than TBP, or as a bridge that holds two VP16 activation domains the correct distance apart to bind TBP and TFIIB simultaneously (Figure 3~). Alternatively, the mediator could be a novel VPlG-binding form of one of the general initiation factors. Is the model shown in Figure 3b appropriate for activators other than GAL4-VP16? Like GAL4-VP16, USF likely achieves antirepression by interacting with TFIID (Meisterernst et al., 1990). In addition, it requires at least one additional factor, which strongly inhibits basal transcription and may interact with human TBP, in order to activate transcription in the absence of nucleosomes (Hoffmann et al., 1990; Meisterernst et al., 1990, 1991). Similarly, the Drosophila activator NTF-1 requires at least one factor tightly bound to TBP, possibly a subunit of native TFIID, in order to activate transcription (Dynlacht et al., 1991). In the case of Spl , its antirepressing effect (Croston et al., 1991) could well reflect an interaction with TFIID. In addition, the N-terminal, nonconserved portion of TBP and at least one factor that associates with TBP seem to be important for activation by Spl in nonchromatin systems (Dynlacht et al., 1991; Meisterernst et al., 1991, and references therein). In at least some of these cases, the activator protein may activate transcription by relieving inhibition caused by factors that associate with TBP (Meisterernst et al., 1991). More generally, recent and continuing progress in the cloning of cDNAs encoding all the general initiation factors should lead naturally to more precise models for transcriptional activation. References Berger, S. L., Cress, W. D., Cress, A., Triezenberg, ente, L. (1990). Cell 61, 1199-1208. Buratowski, S., Hahn, S., Guarente, 56, 549-561.
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