Ordering Promoter Binding of Class III Transcription Factors TFIIIC1 and TFIIIC2 NETA DEAN' and ARNOLD J

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1988, p. 3017-3025 Vol. 8, No. 8 0270-7306/88/083017-09$02.00/0 Copyright C) 1988, American Society for Microbiology Ordering Promoter Binding of Class III Transcription Factors TFIIIC1 and TFIIIC2 NETA DEAN' AND ARNOLD J. BERK' 2* Molecular Biology Institute' and Department of Microbiology,2 University of California, Los Angeles, California 90024 Received 25 September 1987/Accepted 28 April 1988

The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNAA,g genes. The interaction between these genes and TFIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNApArg gene was preincubated with TFTTTC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIITC2 and these genes must occur prior to that of TFIIIC1 or TFITIB. Once TFIIIC2 was bound, TFITIC1 could bind to the tRNAArg and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNAArg gene. TFIIIB activity bound stably to the complex of both genes with TFTTTC2. These results demonstrate that TFTTTC2 is the first transcription factor to bind to these genes and that TFIITB and TFIIIC1 can then interact in either order to form a preinitiation complex.

Transcription of eucaryotic genes requires that accessory tRNA and VAI genes is very stable. As first demonstrated transcription factors interact with promoter DNA sequence with the 5S RNA gene (5), once assembled, these preinitia- elements to effect promoter recognition by RNA polymer- tion complexes are resistant to dilution, high salt, and ases. For genes transcribed by RNA polymerase III (class challenge by an equivalent promoter (16, 25, 30). This III genes), the study of these transcription factors and the stability has allowed examination of the nature of these promoter elements with which they interact has been made protein-DNA interactions in template competition assays. In possible by the development of cell-free in vitro transcrip- this assay, one gene, when preincubated with extracts or tion systems (4, 7, 23, 31, 34; for review, see reference 11). protein fractions, will stably interact with the limiting titra- Most class III genes are distinguished by internal promot- table factor, thereby excluding the transcription of a second ers within the coding sequence. Mutational analyses have gene added subsequently. By controlling conditions so that precisely defined those DNA sequences required for pro- each of the different components required for transcription is moter function. In the case of the adenovirus VAI RNA and made limiting, the nature of the interaction between class III tRNA genes, these consist of two regions, each about 11 genes and class III transcription factors has been examined. base pairs in length. The first region is termed the A block or These assays have demonstrated that there is a precisely 5' internal control region centered at + 13 and +23 for the defined order with which these proteins interact with their tRNAArg and VAI genes, respectively. The second region is cognate genes. An interaction with TFIIIC is necessary and termed the B block or 3' internal control region, centered at sufficient to form a stable VAI RNA gene complex, in that +56 and +66 for the tRNAArg and VAI genes, respectively preincubation of the VAI RNA gene with TFIIIC only is (15, 18, 20, 32). While both of these promoter elements are sufficient to exclude the transcription of a second gene added highly homologous among the VA and tRNA genes (18), it subsequently (25). From the analysis of a variety of tRNA appears that an intact B block is most critical for promoter genes, it is evident that the stability of the TFIIIC-DNA function (1, 37). Sequences flanking the genes can modulate interaction is gene dependent (1). For some tRNA genes, transcriptional efficiency (30, 33, 35) and may serve to while the association with TFIIIC is the first step in complex regulate the expression of these genes (26, 30, 35). The formation, the complex is greatly stabilized by the presence spacing constraints between these elements do not appear to of TFIIIB (8, 25). The copurification of an activity which be very rigid (2, 9, 12). Insertions of up to 79 base pairs binds to the A and B blocks demonstrated by the method of between these two sequences can be tolerated in the VAI DNase I footprinting assays (17), with an activity that is gene without greatly affecting transcriptional efficiency, but required for template commitment (10, 16, 29, 36), has led to insertions of 88 base pairs or more reduce transcription the model that the significantly. Deletions which shorten the intervening spacer sequence-specific interaction between to than TFIIIC and the A and B blocks is the first step in the less 29 base pairs cannot be tolerated (9). formation of the preinitiation complex. This occurs prior to For the adenovirus VAI and the tRNA genes, at least two the association of TFIIIB, which is thought to be associated chromatographically separable fractions, in addition to RNA in the complex through protein-protein interactions (24). polymerase III (polIII), are required to reconstitute tran- It was recently observed that in the mammalian system the scription in vitro (7, 16, 23, 31). These fractions contain the activity previously designated TFIIIC can be separated into general transcription factors designated TFIIIB and TFIIIC. at least two functional components, both of which are The complex formed between these proteins and the required for transcription. These have been termed TFIIIC1 and TFIIIC2 (13, 38). TFIIIC2 binds with high affinity to the * Corresponding author. B block (6) and is required for transcription (38). It is also the 3017 3018 DEAN AND BERK MOL. CELL. BIOL. limiting component in mammalian extracts required for the and suspended in 100 ml of buffer B (50 mM Tris hydrochlo- formation of stable transcriptional complexes on both the ride [pH 7.9], 25% glycerol, 0.1 mM EDTA, 2 mM dithio- VAI and tRNA genes in vitro (13). The precise function of threitol) plus 5 mM MgCl2. Ammonium sulfate (4.0 M) was TFIIIC1 during transcription is unclear. Although no DNA- added slowly to a final concentration of 0.3 M. Cells were binding activity can be demonstrated alone, when TFIIIC1 is lysed by sonication (20 bursts of 10 s each), with the added to TFIIIC2, both the A and B blocks are protected temperature of the extract maintained at 4°C, and lysis was from digestion by DNase I (38). The separation of TFIIIC monitored by microscopy. Cellular debris was pelleted by into these two components has raised the question of how centrifugation for 1 h in a Ti6O rotor at 55,000 rpm and they function in the process of transcriptional initiation. discarded. Nucleic acid in the supernatant was removed by We used template competition assays to examine the adsorption to DEAE-Sephacel (Pharmacia) equilibrated with nature of the interactions that occur between these newly 0.3 M ammonium sulfate in buffer B, applying 1 mg of identified components of the class III transcriptional machin- nucleic acid per ml of resin. The flowthrough, containing ery. These experiments have defined the order with which pollIl activity, was dialyzed to 0.1 M ammonium sulfate in these components interact with the adenovirus VAI RNA buffer B and applied to heparin-agarose equilibrated at 0.17 and Drosophila melanogaster tRNAArg genes. Through M ammonium sulfate in buffer B, at 10 mg of protein per ml these analyses, we have identified several intermediate spe- of resin. The column was washed with 5 column volumes of cies that formed prior to the fully assembled preinitiation 0.17 M ammonium sulfate in buffer B, and polIl activity was complex. This has led us to propose that for the VAI and eluted with 0.5 M ammonium sulfate in buffer B. Active tRNA genes there are alternative pathways for the formation fractions were pooled, dialyzed to 0.1 M ammonium sulfate of the preinitiation complex. in buffer B, and applied to DEAE-Sephadex at 0.5 mg of protein per ml of resin. This column was developed with a MATERIALS AND METHODS linear gradient (5 column volumes) from 0.1 to 0.5 M Preparation and fractionation of cell extracts. Nuclear ammonium sulfate in buffer B plus 0.2 mg of bovine serum extracts (14) were prepared from 293 cells (19), a human albumin (BSA) per ml. The peak of polIlI activity eluted at embryonic kidney cell line transformed by adenovirus type 250 mM ammonium sulfate. Active fractions were pooled, 5, which constitutively express the viral ElA and E1B dialyzed to 50 mM ammonium sulfate in buffer B, and genes. These were grown in suspension culture in minimal applied to a 1-ml phosphocellulose column. After being essential medium (SMEM, GIBCO) with 5% newborn calf washed with 5 column volumes of 50 mM ammonium sulfate serum (GIBCO) and harvested at 8 x 105 cells per ml. in buffer B plus BSA (0.2 mg/ml), polIll activity was The nuclear extract was chromatographed through phos- step-eluted with 250 mM ammonium sulfate in buffer B with phocellulose for preparation of the B fraction (the 0.35 M BSA (0.2 mg/ml). Active fractions were dialyzed to 200 mM KCl eluate containing TFIIIB and polIll) and the C fraction KCI in buffer A, quick-frozen on dry ice, and stored at (the 0.6 M KCl eluate containing TFIIIC activity) (14). The -70°C. Activity was assayed as described below. B fraction was subsequently rechromatographed through TFIIIC1 was prepared by fast protein liquid chromatogra- phosphocellulose equilibrated at 0.1 M KCl and eluted with phy of the phosphocellulose C fraction on MonoQ Mono- a linear gradient (5 column volumes) from 0.1 to 0.4 M KCl beads (Pharmacia), as described (38). TFIIIC2 was prepared in buffet A (13). Fractions were assayed for the ability to by oligonucleotide sequence-specific DNA affinity chroma- reconstitute transcription when supplemented with TFIIIC1 tography as described (13). While none of these fractions and TFIIIC2. Fractions with peak activity eluted near 0.3 M were homogeneous proteins, the level of cross contamina- KCl and contained TFIIIB and polIlI activity but no detect- tion in these preparations was low enough to require the able TFIIIC1 or TFIIIC2 activity. This B fraction was used presence of four fractions (pollll, TFIIIB, TFIIIC2, and in some experiments (as indicated) to supply both TFIIIB TFIIIC1) to reconstitute transcription under the conditions and polIl activity. For the preparation of TFIIIB free of described below. pollIl activity (10), the phosphocellulose B fraction was Plasmid DNA. pArg (32) contains a 535-base-pair (bp) dialyzed to 50 mM KCl in buffer A and chromatographed HindIII fragment containing the Drosophila melanogaster through DEAE-cellulose (DE52; Whatman) equilibrated at tRNAArg gene inserted into the HindIll site of pBR322. 50 mM KCl in buffer A. TFIIIB activity was eluted with a pVAI contains the pVA (20) Sall-BalI fragment, containing linear gradient (5 column volumes) from 50 to 400 mM KCl nucleotides 10589 to 10810 of the adenovirus type 2 DNA in buffer A. Both TFIIIB and polIlI activity eluted at about sequence, including the VAI RNA gene, inserted into the 150 to 200 mM KCl, although the peak of TFIIIB activity Sall-Smal site of pUC18. eluted at a slightly lower salt concentration than the peak of In vitro transcription reactions. Specific in vitro transcrip- polIII activity. Fractions containing TFIIIB activity from the tion reactions were carried out with 0.8 to 1.2 nM pVAI leading edge were concentrated threefold by dialysis against template and 0.6 nM pArg template. Salt and buffer condi- 20% polyethylene glycol 8000 in buffer A with 200 mM KCI, tions, including that of added protein fractions, were 75 mM followed by dialysis into 200 mM KCl in buffer A plus 10% KCI, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2- glycerol. A 0.5-ml amount ofthis fraction was loaded onto an ethanesulfonic acid)-KOH, pH 7.9, 5 mM MgCl2, 10% 11-ml 15 to 30% glycerol gradient, with a 0.5-ml 75% glycerol glycerol, 1 ,ug of ot-amanitin per ml, 8.75 ,ug of creatine cushion in buffer A. Gradients were sedimented for 24 h at phosphokinase per ml, and 5 mM phosphocreatine. Parallel 39,000 rpm in an SW4OTi rotor. Fractions sedimenting with preincubations were carried out in 20- to 30-,I volumes. an s2o,w value of approximately 3.5 contained TFIIIB activ- Final reaction volumes after the mixing of parallel preincu- ity, as assayed by the ability to reconstitute transcription bations and addition of nucleoside triphosphates (NTPs) are when complemented with TFIIIC1, TFIIIC2, and purified indicated in the figure legends. Transcription reaction con- pollIl, but no detectable polIII activity, as assayed in a ditions under which a particular factor was limiting for nonspecific transcription assay, described below. transcription were determined empirically, and the amounts For the preparation of pollll, 293 cells (8 x 109) were of protein used in reactions are indicated in the figure pelleted, washed twice with cold phosphate-buffered saline, legends. NTPs were added to 500 ,uM each ATP, CTP, and VOL. 8, 1988 PROMOTER-BINDING ORDER OF TFIIIC1 AND TFIIIC2 3019

UTP, 10 ,uM GTP, and 0.05 p.Ci of [a-32P]GTP (Amersham; specific activity, 3,000 Ci/mmol) per to the reaction at the t.l TFPIIC2+l+ times indicated in the figure legends. Transcription reactions were carried out at 25°C for the indicated times. RNA TRIC,l + products were phenol-chloroform extracted and ethanol precipitated prior to electrophoresis on 8% polyacrylamide- TFi,!BC 7 M urea gels and were detected by autoradiography. RNA synthesized nonspecifically by polll from a poly(dA. dT) template was monitored as described (22). Salt and buffer conditions, including that of protein frac- PNAR _ * tions, were 100 to 150 mM ammonium sulfate, 25 mM Tris hydrochloride (pH 7.9) (for protein fractions in buffer A, 100 to 150 mM KCl and 20 mM HEPES-KOH, pH 7.9, was used); 2 mM MnCl2, 500 ,uM ATP, 10 ,uM UTP, 0.05 ,uCi of [a-32P]UTP (Amersham; specific activity, 3,000 Ci/mmol) A g per,ul, 0.02 p.g of poly(dA dT) (Pharmacia) per p.l, and 1 ,ug of a-amanitin per ml. Reaction volumes were 50 p.l, and reactions were carried out for 10 min at room temperature and quenched by removing 40 p.1 and spotting it on DEAE- cellulose filters (Whatman DE81). The filters were washed in 5% dibasic sodium phosphate (five 5-min washes, with H20 rinses in between), followed by a 5-min wash in H20. Filters Lae 2 3 4 5 were then rinsed with 95% ethanol and dried, and radiola- FIG. 1. Minimum of four factors are required for transcription of beled RNA was quantitated by Cerenkov counting. One unit both the VAI and tRNA"g genes in vitro. In vitro transcription of activity represented the incorporation of 1 pmol of UMP reactions were performed for 2 h, with salt conditions as described into RNA in 10 min. in Materials and Methods, and reaction mixes contained both pVAI (3.1 ng/rll) and pArg (2.5 ng/>ll) DNA as templates. Fractions RESULTS included or omitted in each reaction are indicated by + or -, respectively, above each lane. When present in the reaction mix, Interaction with TFIIIC2 is necessary and sufficient for proteins were added at the following concentrations: polIll, 100 U/ template commitment of both the VAI and tRNAArg genes. ml; TFIIIC2, 5 ,ug/ml; TFIIIC1, 75 ,g/ml; and TFIIIB, 100 Rg/ml. The formation of stable transcription complexes can be Products of the transcription reactions were resolved by gel elec- demonstrated by template competition assays. In this assay, trophoresis and detected by autoradiography. Arrows indicate the preincubation of one gene with extracts or fractions will positions of VAI RNA and tRNA"g synthesized in the fully exclude the transcription of a second gene added subse- reconstituted reaction (lane 1) or with either polIIl (lane 2), TFIIIC2 quently by sequestering limiting titratable factors by virtue (lane 3), TFIIIC1 (lane 4), or TFIIIB (lane 5) omitted. of stable interaction with the first template during the first preincubation. We used this assay to analyze the process of Preincubation with the TFIIIC2 fraction alone was suffi- preinitiation complex assembly in the system reconstituted cient for template commitment. When either gene was with TFIIIC1, TFIIIC2, TFIIIB, and polIl. Transcription of preincubated with TFIIIC2 alone, transcription of the sec- the VAI and tRNAArg genes required all four of these protein ond gene was almost entirely excluded (Fig. 2B, lanes 9 and fractions (Fig. 1). These results demonstrate that the chro- 10; Fig. 2C, lanes 2 and 3). Although prior incubation with matographic separation of TFIIIC2 and TFIIIC1 resulted in TFIIIC2 was clearly sufficient for template commitment, in proteins that were functionally distinct from either TFIIIB or the absence of TFIIIC1, TFIIIB, and polIll, a low level of pollIl. As template commitment is due to an activity found transcription of the second template could be detected (Fig. in the C fraction (16, 25, 33), likely candidates for this 2B, lanes 9 and 10; Fig. 2C, lanes 2 and 3). In experiments in activity were either TFIIIC1 or TFIIIC2 or both. which the first template was incubated with TFIIIC2 and To determine which of these factors was required for TFIIIC1 (Fig. 2B, lanes 7 and 8), there was less transcription template commitment, the protocol diagrammed in Fig. 2A of the second template than in experiments in which the first was used. With the VAI and tRNAArg genes used as com- template was incubated with TFIIIC2 alone (Fig. 2B, lanes 9 petitor and reference templates, each competitor gene was and 10). From this result, it appears that the presence of separately preincubated with a subset of components, under TFIIIC1 during the preincubation increased the stability of conditions of excess template, while the remaining factors the TFIIIC2-DNA complex. were either preincubated with the reference gene or added To determine whether the TFIIIC1 and B fractions could along with the NTPs after the preincubation. When both associate stably with the template in the absence of TFIIIC2, templates were present in a preincubation with the full the experiment shown in Fig. 3A was performed. Either the complement of factors, both templates were transcribed tRNA Irg gene (Fig. 3B, lane 2) or the VAI gene (Fig. 3B, (Fig. 2B and C, lane 1), although not equivalently. In these lane 3) was preincubated with TFIIIC1 and B fractions for 25 experiments, a 1.3- to 2-fold molar excess of pVAI template min prior to addition of the reference template. TFIIIC2 was over pArg was used to observe similar levels of RNA then added along with NTPs, and transcription proceeded synthesis. As VAI RNA is roughly twice the length of for 90 min. There was no detectable evidence for preferential tRNA , the rate of initiation from the tRNA promoter transcription of the gene preincubated with the TFIIIC1 and was about two- to fourfold greater than that from the VAI B fractions. The level of VAI or tRNA synthesized under promoter. When either the VAI or tRNAArg gene was these conditions was indistinguishable from that synthesized preincubated with all the fractions prior to the addition of a when both templates were present together during the pre- second gene, transcription occurred preferentially from the incubation with TFIIIC1 and B fractions (Fig. 3B, lanes 1 gene present in the preincubation (Fig. 2B, lanes 5 and 6). and 4). Similar results were observed even when preincuba- 3020 DEAN AND BERK MOL. CELL. BIOL.

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...- - S . ..' n a. S - 8 2 S ^, i.E FIG. 2. Interaction of the VAI RNA or tRNA'rg gene with TFIIIC2 is sufficient for template commitment. (A) Experimental protocol. The protein fractions and DNA included in each preincubation are listed in panels B and C, under tube 1 or tube 2. The templates used in each preincubation were pVAI (V) or pArg (A). These were preincubated with protein fractions in parallel for 25 min, after which they were mixed. NTPs were added with (+) or without (-) B fraction (containing TFIIIB and polIll) as indicated at the right. Cl, C2, and B refer to protein fractions containing TFIIIC1, TFIIIC2, and TFIIIB plus polIII, respectively, and were added at 5, 0.2, and 4 ,ug, respectively, per reaction. After addition of NTPs, reactions were allowed to proceed for 60 min. pVAI was present at 2.2 and 1.8 ng/,ul for panels B and C, respectively. pArg was present at 2.5 ng/,ul in both panels B and C. Reaction conditions are as described in Materials and Methods. tion of template I with the TFIIIC1 and B fractions was been preincubated for the same length of time with TFIIIC2 continued for 90 min (data not shown). From these results and TFIIIC1. In this assay, limiting the quantity of TFIIIB in we conclude that in the absence of TFIIIC2, TFIIIC1 and the reaction was critical, since both templates had been TFIIIB cannot associate stably with the VAI or tRNAArg separately preincubated with TFIIIC2 and -C1 and thereby gene. Consequently, the interaction of these genes with had the potential to be transcribed. Preferential transcription TFIIIC2 was not only sufficient for template commitment, of the gene which was incubated in the presence of TFIIIB but necessary as well. Furthermore, these results imply that reflects stable interaction of TFIIIB with the complex during the TFIIIC2-DNA complex must form prior to the assembly the preincubation. If no such stable interaction occurred and of TFIIIC1 or TFIIIB into the complex. TFIIIB remained in solution, it would be free to interact with TFIIIB is stably sequestered by both the VAI and tRNAArg both templates, resulting in transcription of both templates. genes in the absence of TFHIC1. The role of TFIIIB in the As has been shown for a 5S RNA gene (3), the time required process of transcription is poorly understood. The stable for B to stably associate with these genes was relatively association of TFIIIB with the transcription complex has long. Approximately 60 min was required for TFIIIB to been clearly demonstrated for the 5S RNA gene (3), and associate with the VAI gene and 40 to 60 min was required several studies have suggested that this occurs for the VAI for the tRNAArg gene (data not shown) at the protein and and tRNAArg genes (7, 10, 25) as well. In an attempt to assign DNA concentrations used here. a functional role for TFIIIC1 other than its requirement for Once the time required for stable TFIIIB sequestration transcription, we were interested in determining whether had been determined, the dependency of TFIIIC1 on this TFIIIC1 might function in sequestering TFIIIB onto the interaction was examined by assessing whether TFIIIB preinitiation complex. could be sequestered by either of these genes in the absence Initial experiments were conducted to determine the ki- of TFIIIC1 (Fig. 4A). The VAI or tRNAArg gene, TFIIIC2, netics by which TFIIIB is sequestered by the VAI and and B (limiting; containing TFIIIB and polIII) fractions were tRNAArg genes when all components required for transcrip- preincubated in either the presence or absence of the tion were present. This was done by varying the time of TFIIIC1 fraction. After 80 min, a time sufficient for TFIIIB preincubation of one gene with TFIIIC2, TFIIIC1, and B to be sequestered, the second gene, which had been prein- fractions prior to addition of the second gene, which had cubated with TFIIIC2 (with or without TFIIIC1), was added. VOL. 8, 1988 PROMOTER-BINDING ORDER OF TFIIIC1 AND TF1IIC2 3021

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FIG. 3. TFIIIC1 and TFIIIB cannot assemble onto the VAI or FIG. 4. TFIIIB is stably sequestered by the VAI and tRNA"g tRNAArg gene prior to assembly of the TFIIIC2-DNA complex. (A) genes in the absence of TFIIIC1. (A) Experimental protocol. The Experimental protocol. Either pVAI (V) or pArg (A) was incubated factors and DNA template included during the preincubation are with TFIIIC1 and the B fraction (containing TFIIIB and polII). listed under tube 1 or tube 2, indicated in panels B and C. The After 25 min, the second template was added, and then TFIIIC2 and preincubations were carried out for 80 min, after which NTPs were NTPs were added, and the reaction proceeded for 90 min. Abbre- added with (+) or without (-) other factors. The reaction was then viations are as in Fig. 2. (B) Protein fractions and DNA included in allowed to proceed for an additional 60 min. Salt and buffer each preincubation. TFIIIC1, TFIIIC2, and B fractions were added concentrations are as described in Materials and Methods for at 4.0, 0.4, and 4.0 p.g per reaction, respectively. Preincubations transcription reaction conditions. (B) Protein fractions added were 5 were carried out in 25-pIl volumes, with final reaction volumes (after p.g of TFIIIC1, 0.2 p.g of TFIIIC2, and 2 p.g of B fraction (containing addition of second template, TFIIIC2, and NTPs) of 55 ,ul. pVAI TFIIIB and polIll) per 20-pI preincubation reaction mix, conditions and pArg were added to 2.1 and 2.3 p.g/ml, respectively. Lanes S and in which B was limiting for transcription. The addition of NTPs 6 are controls in which TFIIIC2 (lane 5) or TFIIIC1 (lane 6) was (with or without TFIIIC1) was in a 10-pul volume. Final protein omitted from the reactions. concentrations for the 50-pI reaction mixes (after mixing of tubes 1 and 2 and NTP with or without TFIIIC1 additions) were 0.2 pg of TFIIIC1, 8.0 ng of TFIIIC2, and 40 ng of B fraction (containing both NTPs (and TFIIIC1 for those reactions in which it was TFIIIB and pollIl activity) per pul. pVAI and pArg were at 2.3 and 2.5 ng/,pl, respectively. (C) Protein fractions added were 0.2 pug of were was to pro- absent) added, and transcription allowed TFIIIC2 and 4 pug of TFIIIB (free of pollIl) in 20-,ul preincubation ceed for 60 min. reactions. After tubes 1 and 2 were mixed, 10 p.g of TFIIIC1 and 10 or As described above, when either the VAI tRNAArg U of pollll were added with NTPs in 23 p,1. conditions in which gene was preincubated with the TFIIIC1, TFIIIC2, and B TFIIIB was limiting for transcription. Final protein concentrations fractions for 80 min and then added to the second template, for the 63-RlI reaction mixes were 0.16 pg of TFIIIC1, 6.3 ng of which had been preincubated with TFIIIC2 and TFIIIC1 TFIIIC2, 63 ng of TFIIIB, and 0.16 U of polIlI per pRI. Abbreviations fractions, transcription was predominantly restricted to the are as in Fig. 2. template that had been preincubated in the presence of TFIIIB (Fig. 4B, lanes 1 and 3). This demonstrated that a stable association of TFIIIB occurred with both the VAI and level of RNA synthesis was indistinguishable whether or not tRNAArg genes in the presence of TFIIIC2 and TFI1IC1. TFIIIC1 was present during the preincubation. Adding more B fraction resulted in increased transcription, It should be pointed out that in this experiment, a phos- indicating that some factor in the B fraction was limiting for phocellulose B fraction was used as a convenient source of transcription (data not shown). both TFIIIB and pollIl activities. While the recycling of If TFIIIC1 is required to sequester TFIIIB or a factor in pollIl during transcription of the 5S RNA gene has been the B fraction into the TFIIIC2-DNA complex, then omis- demonstrated (3), these results could not rule out the possi- sion of TFIIIC1 during the preincubations should result in bility that pollII, rather than TFIIIB activity, was seques- the transcription of both templates. We observed that pref- tered on these genes. To address this question, either the erential transcription of the gene incubated in the presence VAI or tRNA gene was preincubated with TFIIIC2 and a of limiting TFIIIB occurred in the case of both the VAI gene more highly purified TFIIIB fraction that was free of pollIl (Fig. 4B, lane 4) and tRNAArg gene (Fig. 4B, lane 2), and the activity (see Materials and Methods and Fig. 1) under 3022 DEAN AND BERK MOL. CELL. BIOL. conditions in which TFIIIB was limiting. After 80 min, these A were added to the second template, which had been incubated with TFIIIC2 alone. NTPs, TFIIIC1, and purified pollll were then added, and transcription proceeded for 60 min. The results of this experiment are shown in Fig. 4C. Lane 1 shows the level of VAI and tRNAArg synthesized when both genes were present during the preincubation. Even in the absence of both TFIIIC1 and polIll during the preincubation, inclusion of TFIIIB during the preincubation B with either the VAI (Fig. 4C, lane 2) or tRNAArg (Fig. 4C, lane 3) gene resulted in their preferential transcription. These results demonstrate that TFIIIB can be sequestered onto the complex of TFIIIC2 with both the VAI and tRNAArg genes in the presence or absence of TFIIIC1 and pollll. From this it can be inferred that the association of I I TFIIIB in the complex occurred via a protein-protein interaction with TFIIIC2 or an interaction with the TFIIIC2- DNA complex. Furthermore, this interaction occurred independently of TFIIIC1, which indicated that TFIIIC1 is not required for the assembly of TFIIIB on the TF1IIC2-DNA complex. TFIIICI is sequestered differently by the VAI and tRNAArg genes. The FIG. 5. TFIIIC1 is sequestered differently by the VAI and results demonstrating that TFIIIB could be stably tRNAArg genes. (A) Experimental approach. The protein factors and sequestered by both the VAI and tRNAArg genes in the DNA templates included in each preincubation are listed under tube absence of TFIIIC1 left open the question of whether 1 or tube 2 in panel B. In this experiment, equal molar amounts of TFIIIC1 was stably associated with the preinitiation tran- each template were used. In each preincubation, 2 ,ug of TFIIIC1, scription complex. To answer this question, the experimen- 0.3 ,ug of TFIIIC2, and 5 ig of B fraction (containing TFIIIB and tal protocol diagrammed in Fig. 5A was carried out. As in the polIl) in 20-,u preincubation reaction volumes were determined to approach described above for determining TFIIIB seques- be conditions in which TFIIIC1 was limiting for transcription. Final tration, one template was preincubated with fractions con- protein concentrations, after tubes 1 and 2 were mixed, were 2 jg of taining TFIIIB and pollIl, TFIIIC2, and TFIIIC1 fractions TFIIIC1, 0.6 ,ug of TFIIIC2, and 10 ,ug of B fraction in a 50-,u under reaction. Preincubations were carried out for 20 min, and transcrip- conditions in which TFIIIC1 was limiting for tran- tion was allowed to proceed by the addition of NTPs (with or scription, while the other template was preincubated with without B fraction, as indicated in panel B). The addition of NTPs fractions containing TFIIIB, pollIl, and TFIIIC2. If TFIIIC1 (with and without B) was in a 10-,u volume with salt and buffer is stably associated with the preinitiation complex, prefer- conditions as described for final transcription reaction conditions in ential transcription of the gene incubated in the presence of Materials and Methods for 80 min. TFIIIC1 should occur. If, on the other hand, TFIIIC1 is not stably associated in the complex, it would be free to interact with both genes after the preincubation, as both genes were After a 20-min preincubation, these were mixed and fraction in the potentially committed state by virtue of their associ- B was added, along with NTPs. Under these conditions, ation with TFIIIC2 and TFIIIB. Figure 5B (lane 1) shows the preincubation of pArg in the presence of TFIIIC1 resulted in level of RNA synthesized when both genes were present in almost exclusive transcription of tRNAArg (Fig. 5B, lane 4), the preincubation. Note that in this experiment, the level of demonstrating that TFIIIC1 can be sequestered in the ab- VAI RNA synthesized was less than that of tRNAArg. This sence of TFIIIB. The increased level of tRNAArg synthe- was always the case in reactions in which TFIIIC1 was sized over that when both templates were present during the limiting and probably resulted from a higher affinity of preincubation was the same whether or not TFIIIC1 was TFIIIC1 for tRNAArg than for the VAI preinitiation complex present during preincubation. Addition of TFTIIC1 to the (see below). When the tRNAArg gene was preincubated with reactions increased RNA synthesis, indicating that TFIIIC1 the TFIIIC2, TFIIIC1, and B fractions for 20 min and added was limiting (data not shown). These results indicate that to pVAI which had been preincubated in parallel with only TFIIIC1 can be sequestered onto the TF111C2-tRNAArg gene the TFIIIC2 and B fractions, tRNAArg was preferentially intermediate. TFIIIC1 was assembled in the absence of synthesized, demonstrating that TFIIIC1 is stably seques- TFIIIB, but this required TF1IIC2, implying that this inter- tered by this gene (Fig. 5B, lane 2). When pVAI was action occurred via an association with TFTIIC2 or the preincubated with the TFIIIC2, TFIIIC1, and B fractions TF1I1C2-DNA complex. under conditions in which TFIIIC1 was limiting, there was When the VAI gene was preincubated with TFIIIC1 some preferential transcription of the VAI gene. Quantita- (limiting) and TFIIIC2 and added to the tRNAArg gene, tion of these results indicated that VAI RNA synthesis had which had been separately preincubated with TF1IIC2, increased about threefold over that which occurred when TFIIIC1 was poorly sequestered (Fig. SB, lane 5). The level both genes were present during the preincubation, with a of VAI RNA synthesis increased twofold over that observed concomitant decrease in the level of tRNAArg synthesis. when both templates were present together during the pre- To determine whether the prior association of TFIIIB was incubation (Fig. SB, lane 1), indicating that the sequestration required for the sequestration of TFIIIC1, the same experi- of TFIIIC1 by the TF1IIC2-VA1 complex was less efficient in ment was performed but TFIIIB was left out of the prein- the absence of TFIIIB. Even at preincubation times of up to cubation and added just prior to the addition of NTPs. Each 80 min, complete TFIIIC1 sequestration was not observed gene was preincubated with TFTTIC2 and TFIIIC1 (limiting) on the VAI template (data not shown). This was in contrast while the other was preincubated in parallel with TFIIIC2. to the results observed for the tRNAArg gene, for which, VOL. 8, 1988 PROMOTER-BINDING ORDER OF TFIIIC1 AND TFIIIC2 3023 under these conditions, sequestration of both TFIIIB and (27), raising the possibility that TFIIID is functionally ho- TFIIIC1 occurred independently. It appeared that under mologous to either TFIIIC1 or TFIIIC2. In the reconstituted these conditions TFIIlC1 was not bound tightly by the VAI Bombyx mori system, however, no single component is gene, even though TFIIIC2 and TFIIIB were. sufficient for template commitment of the B. mori tRNAAla We interpret these results to indicate that the ability of the gene in a template competition assay. In combination with VAI gene-TFIIIC2 complex to sequester TFIIIC1 is less TFIIID, silkworm-derived TFIIIB or TFIIIC is required for than that of the equivalent complex on the tRNAArg gene. template commitment (27). In contrast, we observed that the While the preferential transcription of the VAI gene (Fig. interaction of TFIIIC2 alone was sufficient for template 5B, lanes 3 and 5) indicated that TFIIIC1 can associate with commitment of the VAI and tRNAArg genes. These differ- the VAI gene complex, the high level of tRNA transcription ences may be the result of both differences between the indicated that this association was relatively unstable. properties of insect and mammalian transcription factors and DNA sequence variation between the genes analyzed. In DISCUSSION fact, examination of the B block in the B. mori tRNAAla gene Much research has focused on the function of TFIIIC reveals a very poor correlation with the consensus B block. during transcriptional initiation. The separation of mamma- The putative B. mori TFIIIC2 homolog may have a low lian TFIIIC into two components (13, 38) prompted the affinity for this degenerate B block, resulting in an interme- examination of how these newly identified components diate which lacks the stability required for template commit- function in the process of transcription. The ordered se- ment. Addition of other factors stabilized the complex quence of interactions leading to the fully assembled tran- sufficiently for template commitment to be observed (27). scription complex on the adenovirus VAI and the D. mela- This may be similar to our observation that complexes nogaster tRNAArg genes was defined by using template formed with TFIIIC1, TFIIIC2, and B fractions excluded competition assays. transcription of the second template more completely than An interaction with one or more components in the did complexes formed with TFIIIC2 alone (Fig. 2B). TFIIIC2 fraction was shown to be both necessary and Preincubation of TFIIIB with the VAI gene- or tRNAArg sufficient for template commitment. When either the VAI or gene-TFIIIC2 complex excluded transcription of a second tRNAAg gene was preincubated with only the TFIIIC2 template (Fig. 4). We interpret these results to indicate that fraction, transcription of the reference template was almost TFIIIB is stably bound in a complex composed of the gene, completely excluded. As determined by DNA-binding anal- TFIIIC2, and TFIIIB. Similarly, preincubation of TFIIIC1 yses, the TFIIIC2 fraction contains a component which with the tRNAArg gene-TFIIIC2 complex preempted the binds with high affinity (6) and specificity (13, 38) to the B transcription of a subsequently added VAI gene. We inter- block, and it is this region of the DNA which has been shown pret this result to indicate that TFIIIC1 is stably bound in a to be most critical for promoter strength and template complex with TFIIIC2 on this promoter. However, more commitment (1, 30, 33, 36). In the following model, we complex models are also consistent with these results. assume that the DNA-binding protein and the protein re- Direct physical characterization of the transcription factors quired for in vitro transcription are the same molecule, and the intermediates in the assembly of the preinitiation which we refer to as TFIIIC2. This assumption was recently complex will be required to confirm and extend these mod- confirmed by purification of TFIIIC2 to homogeneity (S. K. els. Yoshinaga and A. J. Berk, unpublished results). The role of TFIIIC1 in transcription remains unclear. It The results of the template commitment assays (Fig. 2 and has been proposed that TFIIIC1 interacts with the A block 3) demonstrate that the first step in complex formation is the (38). This is based on the observation that, while TFlIIC1 interaction of TFIIIC2 with the B block. As it has previously alone does not exhibit sequence-specific DNA-binding activ- been shown that an interaction with a component in the ity, in the presence of TFIIIC2, binding of both the B and A TFIIIC fraction was necessary and sufficient to form a stable blocks was observed. For convenience, we refer to TFIIIC1 complex with the VAI gene (25) and that the B block was the as a single protein, but TFIIIC1 activity may require more critical promoter element in template competition experi- than one protein in the TFIIIC1 fraction. We observed here ments (9, 16), this result was not surprising. The somewhat that TFIIIC1 exhibited a different affinity towards the VAI unexpected result was that this was also observed with the and tRNAAg genes. A component of the TFIIIC1 fraction Drosophila tRNAArg gene. Earlier studies have demon- was clearly sequestered by the tRNAArg gene following its strated that in fractionated extracts prepared from Droso- interaction with TFIIIC2. In contrast to the situation for the phila cells, both TFIIIC and TFIIIB are required to form a tRNAArg gene, TFIIIC1 was less stably sequestered by the stable complex on the Drosophila tRNA'rg gene (8). These VAI gene, as tested by challenge with the tRNAAg gene. apparently contradictory results may be explained by the Nonetheless, in the absence of a competitor gene, addition demonstration that the homologous Drosophila and human of TFIIIC1 led to greater A block protection from DNase I class III transcription factors are not entirely equivalent. than with the VAI-TFIIIC2 complex alone (38). The lower While the Drosophila C fraction plus the HeLa B fraction affinity of TFIIIC1 for the VAI-TFIIIC2 complex than for can support transcription, the Drosophila B fraction plus the the tRNAAr9-TFIIIC2 complex may reflect DNA sequence HeLa C fraction are incompatible (8). The differences be- differences. Interestingly, after close examination of the A tween Drosophila and human class III transcription factors block region in the VAI RNA gene, the sequence 5'- are further exemplified by differences in the requirement for TGGTCTGGTGG-3' did not conform well to the consensus upstream sequences for transcription in Drosophila cell A block sequence, TGGCNNAGTGG, in that a G residue verses HeLa cell extracts. While flanking regions have been occurred in position 7. An A residue is found at this position shown to modulate transcriptional efficiency in the Droso- in 100% of the more than 80 tDNAs examined by Galli et al. phila system, flanking sequences have much less of an effect (18) and is therefore considered invariant in these promoter on transcription in HeLa cell extracts (30, 32). elements. Indeed, a single G to A transition at this position Recently, the identification of a new class III transcription in a mutant VAI gene increases its transcription over the factor (TFIIID) has been reported in the silkworm system wild type by twofold (28). Consequently, the relatively low 3024 DEAN AND BERK MOL. CELL. BIOL.

TFUB TFUIC1 A B %

TF+C2

A B B

pblm + A B NTP's DNA lTFMCI N.. TRIt

A B RNA FIG. 6. Model for the different possible pathways to the formation of a fully assembled preinitiation complex. "A" and "B" refer to A and B block regions of the internal promoter. affinity with which TFIIIC1 interacts with the TFIIIC2-VAI This work was supported by Public Health Service grant CA41062 gene intermediate may be the result of a poor VAI A block. from the National Cancer Institute. In contrast, the tRNAArg gene contains an A block sequence LITERATURE CITED (5'-TGGCGCAATGG-3') that conforms quite well to the 1. Baker, R. E., and B. D. Hall. 1984. Structural features of yeast consensus sequence, the only deviation being in a position tRNA genes which affect transcription factor binding. EMBO J. which is variant among those tDNAs examined (18). The 3:2793-2800. lower efficiency with which the TFIIIC2-VAI gene interme- 2. Bhat, R. A., B. Metz, and B. Thimmappaya. 1983. Organization diate sequestered TFIIIC1, in contrast to the TFIIIC2- of the noncontiguous promoter components of the adenovirus tRNAArg gene intermediate, is consistent with the model that VAI gene is strikingly similar to that of the eucaryotic tRNAs. TFIIIC1 interacts with the A block. Mol. Cell. Biol. 3:1996-2005. The stability of the various protein-protein or protein- 3. Bieker, J. J., P. L. Martin, and R. G. Roeder. 1985. Formation of a rate limiting intermediate in 5S RNA gene transcription. DNA interactions enabled us to order and identify several of Cell 40:119-127. the intermediates that were formed prior to the fully assem- 4. Birkenmeier, E. H., D. D. Brown, and E. Jordan. 1978. A bled preinitiation complex. This led us to propose the model nuclear extract of Xenopus laevis oocytes that accurately tran- that formation of the class III stable transcription complex scribes 5S RNA genes. Cell 15:1077-1086. on these genes can occur by different possible pathways 5. Bogengahen, D. F., W. M. Wormington, and D. D. Brown. 1982. involving several intermediates. In the model shown in Fig. Stable transcription complexes of Xenopus SS RNA genes: a 6, the preferred pathway for the formation of the fully means to maintaining the differentiated state. Cell 28:413-421. assembled VAI complex would proceed via the sequential 6. Boulanger, P. A., S. K. Yoshinaga, and A. J. Berk. 1987. association of TFIIIC2, followed by TFIIIB, followed by DNA-binding properties and characterization of human transcription factor TFIIIC2. J. Biol. Chem. 262:15098-15105. TFIIIC1. In the case of the tRNAArg gene, after the binding 7. Burke, D. J., J. Schaack, S. Sharp, and D. Soll. 1983. Partial of TFIIIC2, either TFIIIC1 or TFIIIB can be assembled onto purification of Drosophila Kc cell RNA polymerase III tran- the complex with no apparent preference. The kinetic con- scription components. J. Biol. Chem. 258:15224-15231. trol of the overall process will be governed by the rate- 8. Burke, D. J., and D. Soil. 1985. Functional analysis of fraction- limiting step. By analogy with bacterial promoters (21), it ated Drosophila Kc cell tRNA transcription components. J. may be that for different class III promoters, different Biol. Chem. 260:816-823. intermediate steps in transcription complex formation might 9. Cannon, R. E., G. J. Wu, and J. F. Railey. 1986. Functions of be rate limiting, due to DNA sequence variation and the and interactions between the A and B blocks in the adenovirus resulting differences in type 2-specific VAI RNA gene. Proc. Natl. Acad. Sci. USA 83: transcription factor affinity. This may 1285-1289. provide added levels at which regulated expression of these 10. Carey, M. F., S. P. Gerrard, and N. R. Cozzarelli. 1986. genes may be controlled in vivo by regulating the activity of Analysis of RNA polymerase III transcription complexes by gel the different transcription factors involved in the transcrip- filtration. J. Biol. Chem. 261:4309-4317. tion of class III genes. 11. Ciliberto, G., L. Castagnoli, and R. Cortese. 1983. Transcription by RNA polymerase III. Curr. Top. Dev. Biol. 18:59-88. ACKNOWLEDGMENTS 12. Ciliberto, G., C. Traboni, and R. Cortese. 1982. Relationship between the two components of the split promoter of eukaryotic We thank Carol Eng for her fine technical assistance in the growth tRNA genes. Proc. Natl. Acad. Sci. USA 79:1921-1925. of cells used for the preparation of extracts and fractions and are 13. Dean, N., and A. J. Berk. 1987. Separation of TFIIIC into two grateful to our colleagues for their helpful discussions. functional components by sequence specific DNA affinity chro- VOL. 8, 1988 PROMOTER-BINDING ORDER OF TFIIIC1 AND TFIIIC2 3025

matography. Nucleic Acids Res. 15:9895-9907. 27. Ottonello, S., D. H. Rivier, G. M. Doolitle, L. S. Young, and 14. Dignam, J. D., P. L. Martin, and R. G. Roeder. 1983. Eukary- K. U. Sprague. 1987. The properties of a new pol III transcrip- otic gene transcription with purified components. Methods tion factor reveal that transcription complexes can assemble by Enzymol. 101:582-598. more than one pathway. EMBO J. 6:1921-1927. 15. Fowlkes, D. M., and T. Shenk. 1980. Transcriptional control 28. Rohan, R. M., and G. Ketner. 1987. A comprehensive collection regions of the adenovirus VAI RNA gene. Cell 22:405-413. of point mutations in the internal promoter of the adenoviral 16. Fuhrman, S. A., D. R. Engelke, and E. P. Geiduschek. 1984. VAI gene. J. Biol. Chem. 262:8500-8507. HeLa cell RNA polymerase III transcription factors. J. Biol. 29. Ruet, A., S. Camier, W. Smagowicz, A. Sentenac, and P. Chem. 259:1934-1943. Fromageot. 1984. Isolation of a class III transcription factor 17. Galas, D. J., and A. Schmidt. 1978. DNase footprinting: a simple which forms a stable complex with tRNA genes. EMBO J. 3: method for the detection of protein-DNA binding specificity. 343-350. Nucleic Acids Res. 5:3157-3170. 30. Schaack, J., S. Sharp, T. Dingermann, and D. Soll. 1983. 18. Galli, G., H. Hofstetter, and M. L. Birnstiel. 1981. Two con- Transcription of eukaryotic tRNA genes in vitro: formation of served sequence blocks within eukaryotic tRNAs are major stable complexes. J. Biol. Chem. 258:2447-2453. promoter elements. Nature (London) 294:626-631. 31. Segall, J., T. Matsui, and R. G. Roeder. 1980. Multiple factors 19. Graham, F., J. Smiley, W. Russel, and R. Nairn. 1977. Charac- are required for the accurate transcription of purified genes by teristics of a human cell line transformed by DNA from human RNA polymerase III. J. Biol. Chem. 255:11986-11991. adenovirus type 5. J. Gen. Virol. 36:59-72. 32. Sharp, S., D. DeFranco, T. Dingermann, P. Farrell, and D. Soil. 20. Guilfoyle, R., and W. Weinmann. 1981. Control region for 1981. Internal control regions for transcription of eukaryotic adenovirus VA RNA transcription. Proc. Natl. Acad. Sci. USA tRNAs. Proc. Natl. Acad. Sci. USA 78:6657-6661. 78:3378-3382. 33. Sharp, S., T. Dingermann, J. Schaack, D. DeFranco, and D. Soll. 21. Hawley, D. K., T. P. Malan, M. E. Mulligan, and W. R. 1983. Transcription of eukaryotic tRNA genes in vitro: analysis McClure. 1982. Intermediates in the pathway to open complex of control regions using competition assays. J. Biol. Chem. 258: formation, p. 54-68. In R. L. Rodriquez and M. J. Chamberlin 2440-2446. (ed.), Promoters: structure and function. Praeger, New York. 34. Shastry, B. S., S. Y. Ng, and R. G. Roeder. 1982. Multiple 22. Jaehning, J. A., C. C. Stewart, and R. G. Roeder. 1975. factors involved in the transcription of class III genes in DNA-dependent RNA polymerase levels during the response of Xenopus laevis. J. Biol. Chem. 257:12979-12986. human peripheral lymphocytes to phytohemagglutinins. Cell 4: 35. Sprague, K. U., D. Larson, and D. Morton. 1980. 5' Flanking 51-71. sequence signals are required for activity of silkworm alanine 23. Klekamp, M. S., and P. A. Weil. 1982. Specific transcription of tRNA genes in homologous in vitro transcription systems. Cell homologous class III genes in yeast-soluble cell-free extracts. J. 22:171-178. Biol. Chem. 257:8432-8441. 36. Stillman, D. J., A. L. Sivertsen, P. G. Zentner, and E. P. 24. Klekamp, M. S., and P. A. Weil. 1987. Properties of yeast class Geiduschek. 1984. Correlations between transcription of a yeast III gene transcription factor TFIIIB. J. Biol. Chem. 262:7878- tRNA and transcription factor-DNA interaction. J. Biol. Chem. 7883. 259:7955-7962. 25. Lassar, A. B., P. L. Martin, and R. G. Roeder. 1983. Transcrip- 37. Wu, G., J. F. Railey, and R. E. Cannon. 1987. Defining the tion of class III genes: formation of preinitiation complexes. functional domains in the control region of the adenovirus type Science 222:740-748. 2 specific VAI RNA gene. J. Mol. Biol. 194:423-442. 26. Lofquist, A., and S. Sharp. 1986. The 5' flanking sequences of 38. Yoshinaga, S. K., P. Boulanger, and A. J. Berk. 1987. Resolution Drosophila melanogaster tRNAASn genes differentially arrest of human transcription factor TFIIIC into two functional com- RNA polymerase III. J. Biol. Chem. 261:14600-14606. ponents. Proc. Natl. Acad. Sci. USA 84:3585-3589.