48 124 Functions of Activators
wea25324_ch12_314-354.indd Page 324 324 11/25/10 8:08 PM user-f469 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 12 / Transcription Activators in Eukaryotes DNA-binding domain of GAL4 with the DNA-binding domain of a completely unrelated protein, and produce a functional activator. This demonstrates that the transcriptionactivating and DNA-binding domains of GAL4 can operate quite independently. SUMMARY The DNA-binding and transcription- activating domains of activator proteins are independent modules. We can make hybrid proteins with the DNA-binding domain of one protein and the transcription-activating domain of another, and show that the hybrid protein still functions as an activator. 12.4 Functions of Activators In bacteria, the core RNA polymerase is incapable of initiating meaningful transcription, but the RNA polymerase holoenzyme can catalyze basal level transcription. Basal level transcription is frequently insufficient at weak promoters, so cells have activators to boost this basal transcription to higher levels by a process called recruitment. Recruitment leads to the tight binding of RNA polymerase holoenzyme to a promoter. Eukaryotic activators also recruit RNA polymerase to promoters, but not as directly as prokaryotic activators. The eukaryotic activators stimulate binding of general transcription factors and RNA polymerase to a promoter. Figure 12.13 presents two hypotheses to explain this recruitment: (1) the general transcription factors cause a stepwise build-up of a preinitiation complex; or (2) the general transcription factors and other proteins are already bound to the polymerase in a complex called the RNA polymerase II holoenzyme, and the factors and polymerase are recruited together to the promoter. The truth may be a combination of the two hypotheses. In any event, it appears that direct contacts between general transcription factors and activators are necessary for recruitment. (However, as we will see later in this chapter, some activators require other proteins called coactivators to mediate the contact with the general transcription factors.) Which factors do the activators contact? The answer seems to be that many factors can be targets, but the one that was discovered first was TFIID. Recruitment of TFIID In 1990, Keith Stringer, James Ingles, and Jack Greenblatt performed a series of experiments to identify the factor that binds to the acidic transcription-activating domain of the herpesvirus transcription factor VP16. These workers expressed the VP16 transcription-activating domain as a fusion protein with the Staphylococcus aureus protein A, which binds tightly and specifically to immunoglobulin IgG. (b) (a) TFIIH TFIIE TFIIB Other factors (Mediator) Pol II TFIIF Other factors (Mediator) Pol II TFIIF TFIIH TFIIE TFIIB Holoenzyme TBP TBP TATA TATA Figure 12.13 Two models for recruitment of yeast preinitiation complex components. (a) Traditional view of recruitment. This scheme calls for stepwise addition of components of the preinitiation complex, as occurs in vitro. (b) Recruitment of holoenzyme. Here, TBP binds first, then the holoenzyme binds to form the preinitiation complex. (Source: Adapted from Koleske, A.J. and R.A. Young, An RNA polymerase II holoenzyme responsive to activators. Nature 368:466, 1994.) wea25324_ch12_314-354.indd Page 325 11/25/10 8:08 PM user-f469 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile 12.4 Functions of Activators They immobilized the fusion protein (or protein A by itself) on an agarose IgG column and used these as affinity columns to “fish out” proteins that interact with the VP16activating domain. To find out what proteins bind to the VP16-activating domain, they poured HeLa cell nuclear extracts through the columns containing either protein A by itself or the protein A/VP16-activating domain fusion protein. Then they used run-off transcription (Chapter 5) to assay various fractions for ability to transcribe the adenovirus major late locus accurately in vitro. They found that the flow-through from the protein A column still had abundant ability to support transcription, indicating no nonspecific binding of any essential factors to protein A. However, when they tested the flow-through from the protein A/VP16activating domain column they found no transcription activity until they added back the proteins that bound to the column. Thus, some factor or factors essential for in vitro transcription bound to the VP16-activating domain. Stringer and colleagues knew that TFIID was ratelimiting for transcription in their in vitro system, so they suspected that TFIID was the factor that bound to the affinity column. To find out, they depleted a nuclear extract of TFIID by heating it, then added back the material that bound to either the protein A column or the column containing the protein A/VP16-activating domain. Figure 12.14 shows that the material that bound to protein A by itself could not reconstitute the activity of a TFIID-depleted extract, but the material that bound to the protein A/VP16activating domain could. This strongly suggested that TFIID binds to the VP16-activating domain. To check this conclusion, Stringer and colleagues first showed that the material that bound to the VP16-activating domain column behaved just like TFIID on DEAE-cellulose ion-exchange chromatography. Then they assayed the material that bound to the VP16-activating domain column for the ability to substitute for TFIID in a template commitment experiment. In this experiment, they formed preinitiation complexes on one template, then added a second template to see whether it could also be transcribed. Under these experimental conditions, the commitment to transcribe the second template depended on TFIID. These workers found that the material that bound to the VP16-activating domain column could shift commitment to the second template, but the material that bound to the protein A column could not. These, and similar experiments performed with yeast nuclear extracts, provided convincing evidence that TFIID is the important target of the VP16 transcription-activating domain in this experimental system. SUMMARY The acidic transcription-activating domain of the herpesvirus transcription factor VP16 binds to TFIID under affinity chromatography conditions. M – Heated Control pA VP16 – pA VP16 325 Extract Eluate added 536 nt a b c d e f Figure 12.14 Evidence that an acidic activation domain binds TFIID. Stringer and colleagues fractionated a HeLa cell extract by affinity chromatography with a resin containing a fusion protein composed of protein A fused to the VP16-activating domain, or a resin containing just protein A. Then they eluted the proteins bound to each affinity column and tested them for ability to restore in vitro run-off transcription activity to an extract that had been heated to destroy TFIID specifically. Lanes a–c are controls in which the extract had not been heated. Because TFIID was still active, all lanes showed activity. Lanes d–f contained heated extract supplemented with: nothing (2), the eluate from the protein A column (pA), or the eluate from the column that contained the fusion protein composed of protein A and the transcription-activating domain of the VP16 protein (VP16). Only the eluate from the column containing the VP16 fusion protein could replace the missing TFIID and give an accurately initiated run-off transcript with the expected length (536 nt, denoted at right). Thus, TFIID must have bound to the VP16 transcription-activating domain in the affinity column. (Source: Stringer, K.F., C.J. Ingles, and J. Greenblatt, Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature 345 (1990) f. 2, p. 784. Copyright © Macmillan Magazines Ltd.) Recruitment of the Holoenzyme In Chapter 11 we learned that RNA polymerase II can be isolated from eukaryotic cells as a holoenzyme—a complex containing a subset of general transcription factors and other polypeptides. Much of our discussion so far has been based on the assumption that activators recruit general transcription factors one at a time to assemble the preinitiation complex. But it is also possible that activators recruit the holoenzyme as a unit, leaving only a few other proteins to be assembled at the promoter. In fact, there is good evidence that recruitment of the holoenzyme really does occur. In 1994, Anthony Koleske and Richard Young isolated from yeast cells a holoenzyme that contained polymerase II, TFIIB, F, and H, and SRB2, 4, 5, and 6. They went on to demonstrate that this holoenzyme, when supplemented with TBP and TFIIE, could accurately transcribe a template bearing a CYC1 promoter in vitro. Finally, they showed that the activator GAL4-VP16 could activate this transcription. Because the holoenzyme was provided intact, this last finding suggested that the activator recruited the intact holoenzyme to the promoter rather than building it up step by step on the promoter (recall Figure 12.13). wea25324_ch12_314-354.indd Page 326 326 11/25/10 8:08 PM user-f469 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 12 / Transcription Activators in Eukaryotes By 1998, investigators had purified holoenzymes from many different organisms, with varying protein compositions. Some contained most or all of the general transcription factors and many other proteins. Koleske and Young suggested the simplifying assumption that the yeast holoenzyme contains RNA polymerase II, a coactivator complex called Mediator, and all of the general transcription factors except TFIID and TFIIE. In principle, this holoenzyme could be recruited as a preformed unit, or piece by piece. Evidence for Recruitment of the Holoenzyme as a Unit In 1995, Mark Ptashne and colleagues added another strong argument for the holoenzyme recruitment model. They reasoned as follows: If the holoenzyme is recuited as a unit, then interaction between any part of an activator (bound near a promoter) and any part of the holoenzyme should serve to recruit the holoenzyme to the promoter. This protein–protein interaction need not involve the normal transcription-activating domain of the activator, nor the activator’s normal target on a general transcription factor. Instead, any contact between the activator and the holoenzyme should cause activation. On the other hand, if the preinitiation complex must be built up protein by protein, then an abnormal interaction between an activator and a seemingly unimportant member of the holoenzyme should not activate transcription. Ptashne and colleagues took advantage of a chance observation to test these predictions. They had previously isolated a yeast mutant with a point mutation that changed a single amino acid in a holoenzyme protein (GAL11). They named this altered protein GAL11P (for potentiator) because it responded strongly to weak mutant versions of the activator GAL4. Using a combination of biochemical and genetic analysis, they found the source of the potentiation by GAL11P: The alteration in GAL11 caused this protein to bind to a region of the dimerization domain of GAL4, between amino acids 58 and 97. Because GAL11 (or GAL11P) is part of the holoenzyme, this novel association between GAL11P and GAL4 could recruit the holoenzyme to GAL4-responsive promoters, as illustrated in Figure 12.15. We call the association between GAL11P and GAL4 novel Holoenzyme GAL4 GAL11P TFIID UASG TATA Figure 12.15 Model for recruitment of the GAL11P-containing holoenzyme by the dimerization domain of GAL4. The dimerization domain of GAL4 binds (orange arrow) to GAL11P (purple) in the holoenzyme. This causes the holoenzyme, along with TFIID, to bind to the promoter, activating the gene. because the part of GAL11P involved is normally functionally inactive, and the part of GAL4 involved is in the dimerization domain, not the activation domain. It is highly unlikely that any association between these two protein regions occurs normally. To test the hypothesis that the region of GAL4 between amino acids 58 and 97 is responsible for activation by GAL11P, Ptashne and colleagues performed the following experiment. Using gene-cloning techniques, they made a plasmid encoding a fusion protein containing the region between amino acids 58 and 97 of GAL4 and the LexA DNA-binding domain. They introduced this plasmid into yeast cells along with a plasmid encoding either GAL11 or GAL11P, and a plasmid bearing two binding sites for LexA upstream of a GAL1 promoter driving transcription of the E. coli lacZ reporter gene. Figure 12.16 summarizes this experiment and shows the results. The LexA-GAL4(58–97) protein is ineffective as an activator when wild-type GAL11 is in the holoenzyme (Figure 12.16a), but works well as an activator when GAL11P is in the holoenzyme (Figure 12.16b). If activation is really due to interaction between LexAGAL4(58–97) and GAL11P, we would predict that fusing the LexA DNA-binding domain to GAL11 would also cause activation, as illustrated in Figure 12.16c. In fact, this construct did cause activation, in accord with the hypothesis. Here, no novel interaction between LexA-GAL4 and GAL11P was required because LexA and GAL11 were already covalently joined. The simplest explanation for these data is that activation, at least in this system, can operate by recruitment of the holoenzyme, rather than by recruitment of individual general transcription factors. It is possible, but not likely, that GAL11 is a special protein whose recruitment causes the stepwise assembly of a preinitiation complex. But it is much more likely that association between an activator and any component of the holoenzyme can recruit the holoenzyme and thereby cause activation. Ptashne and colleagues conceded that TFIID is an essential part of the preinitiation complex, but is apparently not part of the yeast holoenzyme. They proposed that TFIID might have bound to the promoter cooperatively with the holoenzyme in their experiments. On the other hand, at least two lines of evidence suggest that the holoenzyme is not recruited as a whole. First, David Stillman and colleagues have performed kinetic studies of the binding of various factors to the HO promoter region in yeast. These studies showed that one part of the holoenzyme, Mediator, binds to the promoter earlier in G1 phase than does RNA polymerase II. Thus, the holoenzyme is certainly not binding as a complete unit, at least to this yeast promoter. Second, Roger Kornberg and colleagues reasoned that, if the holenzyme binds as a unit to promoters, one should find all the components of the holoenzyme in roughly equal amounts in cells. They also knew that determining the concentrations of proteins in cells is tricky. One wea25324_ch12_314-354.indd Page 327 11/25/10 8:08 PM user-f469 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile 12.4 Functions of Activators (a) WT cells Holoenzyme inity No aff GAL11 GAL4(58–97) LexA TATA lexA operator (b) lacZ No activation GAL11P cells Holoenzyme GAL4(58–97) GAL11P LexA TATA lexA operator (c) lacZ Activation gal11 cells Holoenzyme GAL11 LexA TATA lexA operator lacZ Activation Figure 12.16 Activation by GAL11P and GAL11-LexA. Ptashne and colleagues transformed cells with a plasmid containing a lexA operator 50 bp upstream of a promoter driving transcription of a lacZ reporter gene, plus the following plasmids: (a) a plasmid encoding amino acids 58–97 of GAL4 coupled to the DNA-binding domain of LexA plus a plasmid encoding wild-type GAL11; (b) a plasmid encoding amino acids 58–97 of GAL4 coupled to the DNA-binding domain of LexA plus a plasmid encoding GAL11P; (c) a plasmid encoding GAL11 coupled to the DNA-binding domain of LexA. They assayed for production of the lacZ product, b-galactosidase. Results: (a) The GAL4(58–97) region did not interact with GAL11, so no activation occurred. (b) The GAL4(58–97) region bound to GAL11P, recruiting the holoenzyme to the promoter, so activation occurred. (c) The LexA-GAL11 fusion protein could bind to the lexA operator, recruiting the holoenzyme to the promoter, so activation occurred. (Source: Adapted from Barberis A., J. Pearlberg, N. Simkovich, S. Farrell, P. Resnagle, C. Bamdad, G. Sigal, and M. Ptashne, with a component of the polymerase II holoenzyme suffices for gene activation. Cell 81:365, 1995.) cannot do it by measuring mRNA levels because of wide variation in posttranscriptional events such as mRNA degradation and nuclear export. Indeed, concentrations of mRNAs and their respective protein products can 327 deviate from expected values by up to 20- or 30-fold. One can separate proteins by two-dimensional gel electrophoresis and determine their concentrations by mass spectrometry (Chapter 24), but that method is not sensitive enough for proteins, such as transcription factors, found in very low concentrations in vivo. So Kornberg and colleagues chose a method that combines high sensitivity and great accuracy. They began by using gene cloning techniques to attach “TAP” tags to the genes encoding seven different components of the polymerase II holoenzyme. These included RNA polymerase II, Mediator, and five general transcription factors. The TAP tag contains a region from Staphylococcus protein A (Chapter 4) that binds to antibodies of the IgG class. Thus, Kornberg and colleagues could dot-blot cell extracts from the yeast strains carrying genes for TAP-tagged proteins, then probe the blots with an antiperoxidase antibody. The TAP tag on a protein on the blot bound to the antibody, which in turn bound to peroxidase added later, which in turn converted a peroxidase substrate to a chemiluminescent product that could be detected photographically (Chapter 5). The intensities of the bands on the film corresponded to the concentration of TAP-tagged proteins on the blots. With serial dilutions of each extract, these band intensities could be converted to concentrations of each protein per cell by comparing them with the results of a blot of known amounts of a standard, GST-TAP. Figure 12.17 shows sample results. It is clear from the wild-type lane with no TAP-tagged proteins that the background of this method is essentially zero, which is important for accuracy of quantification. It is also clear that there is considerably more RNA polymerase II than Med8, one of the subunits of Mediator. Quantification (Figure 12.17b) showed five to six times as much Rpb3 as any of the subunits of Mediator or of TFIIH. Table 12.1 presents a quantification of the amounts of TFIIF, TFIIE, TFIIB, and TFIID, in addition to the proteins considered in Figure 12.17. Again, RNA polymerase was more abundant than any of the other factors, but the four other general transcription factors were more abundant than either Mediator or TFIIH. Because all of the components of the holoenzyme are not found in roughly equal amounts, it is unlikely that the holoenzyme binds to most promoters as a unit. It is still possible, though, that it is recruited to some promoters as a unit. SUMMARY Activation, at least in certain promoters in yeast, appears to function by recruitment of the holoenzyme, rather than by recruitment of individual components of the holoenzyme one at a time. However, other evidence suggests that recruitment of the holoenzyme as a unit is not common.