Transcription-dependent Structural Changes in the C-terminal Domain of Mammalian RNA Polymerase Subunit IIa/o*

The C-terminal domain of mammalian RNA polym- erase subunit IIa consists of 52-tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain is essentially unmodified in RNA polymerase IIA and extensively phosphorylated in RNA polymerase 110. A monoclonal antibody directed against the C-terminal domain was shown by kinetic enzyme-linked immunosorbent assay to have a 10-fold higher reactivity with RNA polymerase IIA than with RNA polymerase 110. The ability of increas- ing concentrations of this monoclonal antibody to inhibit the initiation and elongation phase of transcrip- tion was determined. Although both phases of the transcription reaction were inhibited, a 10-fold higher concentration of antibody was required to inhibit elongation than was required to inhibit initiation. These results support the hypothesis that RNA polymerase IIA, containing an unphosphorylated C-terminal do- main, is involved in the formation of an initiated complex, whereas elongation is catalyzed by RNA polymerase 110, containing a phosphorylated C-terminal do- main. Further indication that the C-terminal domain undergoes a structural change during the transcription cycle results from the observation that this domain is 3-fold more sensitive to clostripain cleavage in the elongation enzyme than in the free enzyme.

lishes that a minimal number of repeats are essential for in uiuo function (Nonet et al., 1987;Allison et al., 1988, Zehring et al., 1988. Mammalian cells contain two forms of RNA polymerase 11, designated 110 and IIA, that differ in the extent of phosphorylation within the C-terminal domain of the largest subunit (Kim and Cadena and Dahmus, 1987). RNA polymerase I10 is heavily phosphorylated relative to RNA polymerase IIA (Dahmus, 1981;Cadena and Dahmus, 1987).
Photoaffinity labeling of RNA polymerase I1 in cell-free transcription systems, utilizing the major late promoter of adenovirus-2 (Bartholomew et al., 1986), and in isolated HeLa nuclei (Cadena and Dahmus, 1987) indicates that elongation is catalyzed primarily by RNA polymerase 110.
The conservation of the C-terminal domain of subunit IIa from yeast to mammals and the lethal effect of mutations contained in the exon encoding this domain provide strong evidence that the C-terminal domain is required for the transcription of at least some essential cellular genes. The mechanism by which the C-terminal domain functions in transcription is, however, unknown. One possibility is that the Cterminal domain interacts with another component of the transcription apparatus, via protein-protein interactions, to help direct and orient RNA polymerase I1 to the start site of transcription (Corden et al., 1985;Allison et al., 1985;Sigler, 1988). Such an interaction could be mediated, at least in part, by phosphorylation of the C-terminal domain (Cadena and Dahmus, 1987). In these studies, a monoclonal antibody directed against the consensus repeat of the C-terminal domain has been used in an effort to define at what step in the transcription reaction the C-terminal domain is involved and as a probe to detect structural changes during transcription. Results suggest that the extent of phosphorylation of the Cterminal domain changes during the transcription cycle and that extensivephosphorylation of the C-terminal domain may be involved in the transition of enzyme from the initiation to the elongation complex.

Materials
High performance liquid chromatography-purified nucleotides were purchased from ICN. Radiolabeled nucleotides were obtained from Amersham. Sarkosyl (N-lauroylsarcosine, sodium salt), heparin, and clostripain were purchased from Sigma.
A stock clostripain solution of 1 mg/ml was prepared by dissolving lyophilized enzyme in 50 mM Pipes,' pH 6.8, 1 mM CaC12, 5 mM 2mercaptoethanol and incubating on ice for 2 h. Mouse myeloma IgM was obtained from Litton Bionetics, Inc. Synthetic peptide containing three copies of the consensus repeat, Tyr-Ser-Pro-Thr-Ser-pro-Ser, coupled to BSA was a generous gift from J. Corden (The Johns Hopkins University).
Preparation on Transcription E.rtract"S100 extract was prepared from HeLa cells by the method of Weil et al. (1979), as modified by Dahmus and Kedinger (1983). The SlOO extract (15 ml) was applied to a 15-ml heparin-Sepharose column equilibrated with buffer A containing 100 mM KC1 (Davison et al., 1983). The column was washed with 2 column volumes of the same buffer, followed by 1 column volume of buffer A containing 0.24 M KCl. RNA polymerase I1 and transcription factors were eluted with 2 column volumes of buffer A containing 0.6 M KCl. The flow-through fractions contain a stimulatory factor that was further purified by DEAE-cellulose (DE22) chromatography as described by Egly et al. (1984). Peak fractions were identified by promoter-dependent assay and stored in liquid nitrogen. A mixture of 5 pl of the 0.6 M KC1 heparin-Sepharose peak fraction and I p1 of DE22-purified flow-through peak fraction was used in subsequent transcription reactions.
Preparation of DNA Templates-The adenovirus-2 TaqI DNA fragment containing the major late promoter (positions -260 to +560) (designated template 1) was prepared and purified by sucrose gradient centrifugation as previously described (Dahmus and Kedinger, 1983). This DNA fragment was digested with Hincll to obtain a fragment from positions -260 to +405 (designated template 2).
Transcription Reactions-Transcription reactions were carried out in three successive steps. Reaction schemes are shown diagrammatically in Fig. 1. The first two steps involved incubation with antibody and the formation of an initiated complex. Initiation assays refer to reactions in which monoclonal antibody was incubated with extract prior to the formation of an initiated complex, whereas elongation assays refer to reactions in which antibody was added after the formation of an initiated complex. The third step was elongation and was initiated by the addition of remaining nucleotides.
For elongation assays, transcription extract (6 pl) was preincubated with 25 ng of DNA template 1 in a 1 2 4 reaction containing 25 mM Tris-HCI, pH 7.9,12.9 mM MgC12, 50 mM KCl, 0.25 mM dithiothreitol, 10% glycerol, 1.25 mM ATP, and 25 pM [a-32P]CTP for 60 min at 30 "c. A 6-pl aliquot of monoclonal antibody (see figure legend for concentrations) was then added, and the reaction was incubated for an additional 60 min at 30 "C. After this addition the concentration of components was identical in both the initiation and elongation assays. A third aliquot (20 pl) and fourth aliquot (12 p1) identical in composition to those described above in the initiation assay were added, and the incubation continued for 60 min at 30 "C.
RNA was purified and analyzed by electrophoresis on a 5% polyacrylamide-urea gel as described in Dahmus and Kedinger (1983). The amount of specific transcript was quantitated by excision of the appropriate bands from the gel, using the autoradiogram as a template, and Cerenkov counting. A section of an unused portion of the gel served as a background.
Preparation of 32P-Labeled RNA Polymerase Zl-RNA polymerase I10 was purified from calf thymus by the method of Kim and Dahmus (1988). Casein kinase I, isolated from calf thymus as described by Dahmus (1981) was further purified by chromatography on Mono S (Pharmacia LKB Biotechnology Inc.). 32P-Labeled RNA polymerase I1 was obtained by incubation of 0.7 pg of RNA polymerase I10 with 3 units of casein kinase I in a 25-pl reaction containing 40 mM Tris-HCl, pH 7.9,80 mM KCl, 6.5 mM MgClz, 0.4 mM dithiothreitol, 16% glycerol, and 10 pM [y"P]ATP (1.76 X lo' cpm/pmol). The reaction mixture was incubated at 37 'C for 10 min.
Photoaffinity Labeling-RNA polymerase I1 was photoaffinity-labeled in a 150-pl reaction under the conditions described by Bartholomew et al. (1986). Photoaffinity-labeled transcription complexes were digested with 75 units of ribonuclease T I (Pharmacia) on ice for 30 min and purified by gel filtration on a 3-ml Sepharose CL-4B (Pharmacia) column. The column was developed with 0.5 X buffer A (Davison et al., 1983) containing 7.5 mM MgC12. Photoaffinity-labeled transcription complexes eluted in the void volume.
Cbstripain Digestion-Aliquots of the casein kinase I labeling reaction (3.5 pI) and purified photoaffinitylabeled RNA polymerase I1 (25 pl) were digested in reactions containing clostripain (concentration indicated in the figure legend) in a final volume of 30 pl. The reaction mixture contained 21 mM Tris-HC1, pH 7.9,41 mM KCI, 6.4 mM MgCl,, 1 mM CaC12, and 1.2 mM ATP. Digestions of phosphorylated RNA polymerase 11 also contained 8.5 pI of heparin-sepharosepurified SI00 HeLa cell extract. Reactions were incubated at 37 "C for 60 min, stopped by the addition of 1.5 lrl of 200 mM EGTA, and denatured in Laemmli sample buffer (Laemmli, 1970).
Polyacrylamide Gel Electrophoresis-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (1970). The resolving gel was a linear gradient of 5-17.5% acrylamide. Gels were silver-stained according to the procedure of Wray et al. (1981), dried, and exposed to x-ray film.

Monoclonal Antibody G7A5 Reacts with the Consensus Re-
peat of the C-terminal Domuin-Monoclonal antibody G7A5 was produced using calf thymus RNA polymerase I1 as immunogen and shown to react with subunits 110 and IIa, but not IIb (Christmann and Dahmus, 1981;Cadena and Dahmus, 1987). Subunits IIa and IIb are products of the same gene and appear to differ only by the absence of the C-terminal domain from subunit IIb (Corden et ul., 1985; Kim and . This result implies the epitope recognized by this monoclonal antibody resides in the C-terminal domain. This was confirmed by determining the reactivity of monoclonal antibody G7A5 with a synthetic peptide containing three copies of the consensus repeat, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This peptide was coupled to BSA, and immunoreactivity was determined by ELISA and protein blotting. The relative affinity as determined by kinetic ELISA (Tsang et  for the synthetic peptide was one-half that determined for RNA polymerase IIA (data not shown).
This monoclonal antibody also reacted with the BSA-coupled synthetic peptide when transferred to nitrocellulose, but did not react with control BSA (data not shown).
Relative Affinity of Monoclonal Antibody G7A5 for RNA Polymerases 110, IIA, and IIB-RNA polymerases 110, IIA, and IIB were purified from calf thymus, and the relative reactivity of monoclonal antibody G7A5 with each subspecies was determined by kinetic ELISA as described under "Experimental Procedures." The concentration of RNA polymerase subspecies was determined by enzymatic assay, assuming a specific activity of 400 units/mg (Kim and . To confirm that wells were coated with equimolar amounts of each subspecies, equivalent units of activity were subjected to SDS-PAGE and stained with Coomassie Blue, and the densitometer scans of RNA polymerases 110, IIA, and IIB were compared. To verify that the retention of RNA polymerases 110, IIA, and IIB was comparable, identically coated wells were also reacted with monoclonal antibody E3E9 directed against the M, = 34,000 subunit. The results summarized in Table I show that the relative affinity of G7A5 for RNA polymerases I10 is less than one-tenth that of RNA polymerase ITA. The fact that phosphorylation of the C-terminal domain reduces affinity by greater than 10-fold suggests that either most of the repeats are phosphorylated or that the phosphorylation of a limited number of repeats results in a major conformational change. Removal of the C-terminal domain (RNA polymerase IIB) reduces the relative affinity of G7A5 for RNA polymerase I1 an additional 10-fold.
The relative reactivity of monoclonal antibody G7A5 for RNA polymerases 110, IIA, and IIB was also confirmed by immunoblotting (data not shown). Equimolar amounts of each subspecies were electrophoresed on SDS-polyacrylamide gels, transferred, and reacted with monoclonal antibody G7A5 or E3E9 as previously described . Under these conditions, G7A5 reacts strongly with subunit IIa, but not to an appreciable extent with either subunit 110 or IIb. Monoclonal antibody E3E9 reacts comparably with the M, = 34,000 subunit of RNA polymerases 110, HA, and IIB.
Effect of Monoclonal Antibody on the Initiation and Elongation Phase of Transcription-One approach to defining the role of the C-terminal domain in transcription is to identify the step(s) in transcription in which it is involved. Since monoclonal antibody G7A5 is known to inhibit promoterdependent transcription (Dahmus and Kedinger, 1983) and to react with the consensus repeat, this antibody provides a useful probe in the analysis of C-terminal domain function. In order to measure the effect of monoclonal antibody G7A5 on initiation, antibody was added in the reaction sequence prior to the formation of an initiated complex, and the effect on transcription was ascertained. The effect of monoclonal antibody on elongation was determined by the addition of antibody after the formation of an initiated complex. "Initiated complex" refers to transcription complexes in which the first phosphodiester bond has been formed. For the adenovirus-2 major late promoter, this requires ATP and CTP. Sarkosyl was added to iimit initiation to a single round (Hawley and Roeder, 1987). The second DNA template, truncated to give a runoff transcript of 405, serves as a control to ensure that Sarkosyl effectively inhibited any new initiation. Reaction sequences are shown schematically in Fig. 1. In order to determine the effect of this monoclonal antibody on the formation of an initiated complex, transcription extract containing RNA polymerase I1 was preincubated with a constant amount of monoclonal antibody containing increasing proportions of polymerase specific antibody. Formation of an initiated complex occurred during the second phase of the reaction following the addition of ATP, CTP, and DNA containing the major late promoter of adenovirus-2, cut to give a runoff transcript of 560 nucleotides. Following the formation of an initiated complex, Sarkosyl was added along with GTP, UTP, and a second DNA template containing the same promoter but truncated to give a NnOff transcript of 405 nucleotides. The amount of transcript produced at the end of the final 60 min incubation was determined as described under "Experimental Procedures. " Fig. 2, lane 2, shows that in the absence of Sarkosyl, both DNA templates were transcribed. The fact that the addition of Sarkosyl, after preincubation with template 1, but before the addition of template 2, abolished the formation of the 405-nucleotide transcript but not the 560-nucleotide transcript indicates that this level of Sarkosyl effectively inhibits initiation (Fig. 2,  lane 3 ) . In this system, we found that 0.08% Sarkosyl completely inhibited new initiation and had only a slight effect on transcription by previously initiated complexes (results not shown).
Reactions in lunes 4-7 contained a constant amount of monoclonal antibody with varying proportions of control and RNA polymerase I1 antibody. The addition of non-polymerase 11 antibody had no effect on the level of transcription (com- pare lanes 3 and 4 ) . In contrast, the addition of RNA polymerase I1 antibody showed a concentration-dependent inhibition of transcription when added prior to initiation (lanes 5-7). Quantitation of several such experiments shows that a concentration of about 300 pg/ml is required for 50% inhibition of the initiation phase of the reaction (Fig. 3). This corresponds closely to the sensitivity found previously (Dahmus and Kedinger, 1983) which indicates that the presence of Sarkosyl does not have an appreciable effect on antibody binding.
In a similar series of reactions, varying concentrations of monoclonal antibody were added after the formation of an initiated complex, and the effect on transcription was ascertained ( Fig. 2, lanes 8-12). Similar to results presented above, the absence of a 405-nucleotide transcript confirms that transcription was limited to a single round. Significant inhibition of transcription was observed only at the highest concentra-  5 and 10, 6 and 11, and 7 and 12 contain 127 pg/ml, 423 pg/ml, and 1270 pg/ml RNA polymerase I1 monoclonal antibody, respectively. In reactions contained in lanes 4-7, antibody was added prior to the formation of an initiated complex (initiation assay). In reactions contained in lanes 9-12, antibody was added after the formation of an initiated complex (elongation assay). Lanes 1 and 13 contain MspI cut and "P-end-labeled pBR322 DNA. The length in nucleotides of the four largest DNA fragments is indicated in the right margin. The position of runoff transcripts from DNA template 1 and 2 is indicated in the left margin.
Log Antibody Concentration   FIG. 3. Quantitation of the effect of monoclonal antibody on initiation and elongation. The amount of the 560-nucleotide transcript was quantitated as described under "Experimental Procedures." The percent activity remaining is equal to the amount of specific transcript produced relative to the amount produced in the presence of control antibody only. The percent activity remaining for initiation phase ( U ) and elongation phase ( U ) assays was plotted as a function of the log of RNA polymerase I1 monoclonal antibody concentration. Schematic representation of the procedure for determining relative sensitivity of the C-terminal domain to clostripain cleavage in the free and elongating enzyme. The conditions for labeling free and elongating enzyme, the purification of the photoaffinity-labeled transcription complex, and the clostripain digestion were as described under "Experimental Procedures." tion of monoclonal antibody (in this case, 1270 pglml). Quantitation of several experiments shows that the antibody concentration required to inhibit elongation is about 10 times higher than that necessary to inhibit initiation (Fig. 3).
Sensitivity of C-terminal Domain to Proteolytic Cleavage in Free and Elongating Enzyme-The fact that RNA polymerase IIA is 10-fold more immunoreactive than RNA polymerase I10 and that the initiation phase of the reaction is 10-fold more sensitive to inhibition by monoclonal antibody G7A5 than is elongation suggests that RNA polymerase IIA is involved in the formation of an initiation complex and that RNA polymerase I10 is involved in elongation. Consequently, the transition of the enzyme from the initiation phase to the elongation phase appears to be associated with phosphorylation of the C-terminal domain. To test the possibility that the transition from initiation to elongation is associated with a conformational change, the relative accessibility of the Cterminal domain in the free and elongating enzyme was determined by measuring its rate of cleavage by the protease clostripain. The experimental protocol is shown diagrammatically in Fig. 4.
Under limiting conditions, clostripain cleaves the C-terminal domain from subunit 110 leaving the remainder of the subunits and the C-terminal domain otherwise intact.' The rate of cleavage of the C-terminal domain was estimated by following the rate of disappearance of radiolabeled subunit

110.
Subunit 110, in the free enzyme, was labeled by phosphorylation with casein kinase I in the presence of [ Y -~~P ] A T P (Dahmus, 1981;Cadena and Dahmus, 1987). Under these conditions, casein kinase I selectively phosphorylates the Cterminal domain of subunit 110 (Cadena and Dahmus, 1987). 32P-Labeled RNA polymerase I1 was digested with increasing concentrations of clostripain in the presence of the transcription extract and analyzed by SDS-polyacrylamide gel electrophoresis. It is apparent from Fig. 5 (lanes 5-9) that increasing concentrations of clostripain resulted in a decrease in the amount of subunit 110. Quantitation of these results from scans of such autoradiographs is shown in Fig. 6.
Since only a small fraction of the RNA polymerase I1 in the extract is transcriptionally active under the conditions of the assay (Dahmus and Kedinger, 1983), the clostripain sensitivity of an elongating enzyme can only be established after selectively labeling the transcriptionally active enzyme. The transcribing enzyme was photoaffinity-labeled as described by Bartholomew et al. (1986). Transcription was carried out and in elongating enzyme ( U ) was quantitated from densitometric scans of autoradiograms from two experiments carried out as described in the legend to Fig. 5. The values were averaged, and the percent subunit 110 remaining relative to subunit 110 present in the control reaction was plotted as a function of the log of clostripain concentration (pg/ml).
in the presence of 4-thio-UTP and [cT-~'P]CTP and the nascent transcript cross-linked to RNA polymerase I1 by irradiation with near UV light. This results in the specific labeling of the two largest subunits, namely 110 and IIc (see Fig. 5, lane 10, and Bartholomew et al., 1986). Transcription complexes containing photoaffinity-labeled RNA polymerase I1 were purified by gel filtration and digested with increasing concentrations of clostripain as described under "Experimental Procedures." Results presented in Fig. 5 show that incubation in the presence of relatively low concentrations of clostripain results in the disappearance of subunit 110 (lanes [11][12][13][14] and the appearance of label in the region of subunit IIb and the free C- terminal domain (lanes 13-16). The concomitant appearance of subunit IIb and free C-terminal do-main with the disappearance of subunit 110 verifies that this range of clostripain concentrations results in the selective cleavage of the C-terminal domain. Quantitation of the disappearance of subunit 110 is shown in Fig. 6. Similar results were obtained when RNA polymerase I10 was photoaffinitylabeled in isolated HeLa nuclei (Cadena and Dahmus, 1987) and digested with increasing concentrations of clo~tripain.~

DISCUSSION
The observation that monoclonal antibody G7A5 reacts with a synthetic peptide containing three copies of the Cterminal domain consensus repeat and reacts with RNA polymerases I10 and IIA but not IIB (Christmann and Dahmus, 1981;Cadena and Dahmus, 1987) establishes that G7A5 recognizes a determinant in the C-terminal domain of subunit IIa. The relative affinity of this monoclonal antibody for RNA polymerases I10 and IIA, as determined by kinetic ELISA, suggests that the epitope recognized is the unmodified repeat. Phosphorylation of the C-terminal domain decreases monoclonal antibody reactivity by a t least 10-fold.
The monoclonal antibody G7A5 inhibits the initiation phase of transcription a t a 10-fold lower concentration than is required to inhibit elongation. The observation that higher concentrations of antibody also inhibit elongation suggests that binding of monoclonal antibody to the C-terminal domain can inhibit both the initiation and elongation phase of transcription. The differential sensitivity of the initiation and elongation phase to inhibition by monoclonal antibody G7A5 is likely the result of differential immunoreactivity brought about by phosphorylation of the C-terminal domain during the transcription cycle. Photoaffinity labeling of RNA polymerase 11, in a cell-free transcription system (Bartholomew et al., 1986) and in isolated HeLa nuclei (Cadena and Dahmus, 1987), establishes that elongation of most class I1 genes is catalyzed by RNA polymerase 110. These studies do not, however, preclude the possibility that another form of RNA polymerase I1 is involved in initiation. Since the relative affinity of monoclonal antibody G7A5 is 10-fold higher for RNA polymerase IIA than for RNA polymerase 110, the increased sensitivity of the initiation phase is consistent with the idea that RNA polymerase IIA is involved in the formation of an initiation complex. These results are in agreement with the model recently proposed in which phosphorylation of the C-terminal domain is thought to lead to the release of enzyme from the initiated complex (Sigler, 1988;Dahmus et al., 1989;. The possibility that the phosphorylation of RNA polymerase I1 may be associated with a conformational change within the C-terminal domain was examined by determining the relative sensitivity of this domain to clostripain cleavage in the free and elongating enzyme. Results of such experiments indicate that the C-terminal domain of subunit 110 in the elongating complex is approximately 3 times more accessible to cleavage by clostripain than in the nontranscribing enzyme. This suggests that the C-terminal domain of subunit 110 in the elongating enzyme may be in a more extended conformation, relative to its conformation prior to initiation. This is also supported by the observation that the sedimentation constant of RNA polymerase I10 is slightly less than that of RNA polymerase IIA (Dahmus, 1981). I t is not possible to conclude from these studies, however, whether or not the conformational change observed results directly from the phosphorylation of the C-terminal domain.
An alternate interpretation of the increased resistance of M. E. Dahmus, unpublished results. elongation to inhibition by monoclonal antibody is that conformational changes, or the association of other factors with the enzyme, mask the determinants recognized by monoclonal antibody G7A5. Although such possibilities cannot be eliminated at this time, the observation that the C-terminal domain of RNA polymerase I10 appears to be in a more extended conformation suggests that this domain is accessible.
In addition to the studies presented here, the idea that RNA polymerase IIA is involved in the formation of an initiation complex and that phosphorylation of the C-terminal domain is involved in the transition to an elongating complex is supported by the following observations. In vitro transcription of the adenovirus-2 major late promoter with purified RNA polymerases I10 and IIA indicates that the rate of transcription catalyzed by RNA polymerase IIA is greater than that of RNA polymerase I10 (Kim and . The subspecies refer to the nature of the input enzyme. The decreased transcriptional activity of RNA polymerase I10 may result from the need to dephosphorylate the C-terminal domain prior to initiation. Secondly, transcription extracts prepared from HeLa cells according to the procedures of Weil et al. (1979), Davison et al. (1983), and Moncollin et al. (1986) contain an activity that catalyzes the conversion of RNA polymerase IIA to IT0.4 Finally, it is important to note that an ATP requirement has previously been demonstrated for an early step in promoter-dependent transcription catalyzed by RNA polymerase I1 (Sawadogo and Roeder, 1984;Conaway and Conaway, 1988). According to the model proposed above, activation of transcription is dependent on phosphorylation of the C-terminal domain of subunit IIa. Consequently, the protein kinase(s) that catalyze this conversion would be an essential transcription factor(s). A critical test of this model requires an identification of the factor(s) responsible for the phosphorylation of the C-terminal domain and a demonstration that such a factor(s) is essential for promoter-dependent transcription. A characterization of the state of phosphorylation of the RNA polymerase I1 as a function of its position in the transcription cycle will also be essential to more precisely define the functional significance of the C-terminal domain of subunit IIa.