©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Activity of COOH-terminal Domain Phosphatase Is Regulated by a Docking Site on RNA Polymerase II and by the General Transcription Factors IIF and IIB (*)

Ross S. Chambers (1), Bo Qing Wang (2), Zachary F. Burton (2), Michael E. Dahmus (1)(§)

From the (1)Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California, Davis, California 95616 and the (2)Department of Biochemistry, Michigan State University, E. Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is supported by the observations that (a) phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possibility that the general transcription factors may be involved in the regulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.


INTRODUCTION

RNA polymerase (RNAP)()II is a multisubunit enzyme responsible for the transcription of protein coding genes in eukaryotes. The largest subunit contains at its COOH terminus a highly conserved domain not found in other RNAPs (for a recent review see Ref. 1). This COOH-terminal domain (CTD) is composed of tandem repeats of the consensus sequence YSPTSPS. RNAP II from plasmodium contains 19 repeats, mammals contain 52 repeats, and other eukaryotes contain an intermediate number of repeats. Genetic studies show that the CTD is essential in vivo although it is dispensable for transcription from some promoters in vitro.

The CTD of mammalian RNAP II contains more than 250 serine/threonine/tyrosine residues and is subject to extensive phosphorylation. RNAP II with a phosphorylated CTD is referred to as RNAP IIO whereas the unphosphorylated form is referred to as RNAP IIA. Phosphorylation of the CTD results in a decrease in the electrophoretic mobility of the largest subunit in SDS-PAGE. The phosphorylated subunit, designated IIo, has an apparent M of 240,000 in mammalian cells whereas the unphosphorylated subunit, designated IIa, has an apparent M of 214,000. Interconversion of the two forms and separation on SDS-PAGE form a simple assay for CTD kinases and phosphatases. A third form, RNAP IIB, which lacks the CTD as a consequence of limited proteolysis during the purification process, is not found in vivo.

A multiplicity of protein kinases capable of phosphorylating the CTD in vitro have been characterized. It is not clear if this multiplicity is a reflection of multiple CTD kinases functioning at the same step in the transcription cycle, discrete CTD kinases functioning at different steps in the transcription cycle, and/or the adventitious use of the CTD as an in vitro substrate by protein kinases that do not phosphorylate the CTD in vivo. Of special interest is the CTD kinase associated with TFIIH(2, 3, 4, 5, 6, 7) and yeast CTK1 (8). The association of CTD kinase with an essential transcription factor suggests that this kinase functions in CTD phosphorylation in vivo. The observation that disruption of the gene encoding the largest subunit of CTK1 results in a change in RNAP II phosphorylation suggests that CTK1 is also one of several protein kinases that influence phosphorylation of the CTD in vivo.

Much less is known about CTD phosphatases. A phosphatase that can dephosphorylate RNAP IIO has been purified from a HeLa cell extract (9). This enzyme is a type 2C phosphatase, requires Mg for activity, and has a molecular weight of about 200,000. The CTD phosphatase is specific for the dephosphorylation of serine and threonine in the consensus CTD repeat in that it does not dephosphorylate RNAP II phosphorylated by casein kinase II on the most COOH-terminal serine adjacent to the consensus repeat. Furthermore, CTD phosphatase does not dephosphorylate RNAP IIO prepared in vitro by phosphorylation of RNAP IIA with c-Abl tyrosine kinase.() CTD phosphatase can dephosphorylate RNAP IIO purified from calf thymus or RNAP IIO prepared in vitro by the phosphorylation of RNAP IIA with two different HeLa cell CTD kinases. Interestingly, CTD phosphatase and several CTD kinases appear to act processively resulting in the complete conversion of RNAP IIO to IIA and RNAP IIA to IIO, respectively.

Results from a variety of studies indicate that RNAPs IIA and IIO have distinct functions in transcription and that each round of transcription is associated with the reversible phosphorylation of the CTD. Promoter-dependent transcription in vitro requires a set of general transcription factors including TFIIA, -B, -D, -E, -F, -H, and -J (for a recent review see Ref. 10. TFIIA, -B, and -D assemble onto the promoter followed by RNAP II which is recruited as a complex with TFIIF. Finally TFIIE, -H, and -J assemble into the preinitiation complex. The state of phosphorylation of the CTD appears to play a direct role in the recruitment of RNAP as indicated by the observation that RNAP IIA is preferentially recruited relative to RNAP IIO to both the adenovirus-2 major late (Ad2-ML) and dihydrofolate reductase promoters(11, 12, 13) . Presumably the CTD kinase associated with TFIIH phosphorylates the CTD immediately preceding or concomitant with the initiation of transcription. The functional consequences of phosphorylation of the CTD are not known but likely alter interactions mediated by the unphosphorylated CTD in the preinitiation complex. Phosphorylation of the CTD appears dispensable for transcription in vitro from the Ad2-MLP(14) .

Transcript elongation is catalyzed by RNAP IIO. Recently, it has been shown that RNAP II paused early in elongation on some Drosophila genes is unphosphorylated(15) . The observation that activation of transcription correlates with phosphorylation of the CTD suggests that CTD phosphorylation may be essential to generate an elongation competent form of RNAP II. Accordingly, the CTD may play a positive role in transcript elongation. The CTD is presumably dephosphorylated by CTD phosphatase after completion of the transcript.

Since RNAPs IIA and IIO have distinct roles in transcription, CTD kinases and CTD phosphatase(s) that catalyze the interconversion of these two forms must be carefully regulated. Both CTD kinases and CTD phosphatases have the potential to be positive or negative effectors of transcription depending on where they act in the transcription cycle. For example, CTD phosphatase that dephosphorylates free RNAP IIO may function as a positive effector of transcription by increasing the amount of RNAP IIA available for recruitment to the preinitiation complex. Conversely, CTD phosphatase that dephosphorylates RNAP IIO in an elongation complex could in principle act as a negative effector. This is based on the assumption that the phosphorylated CTD plays a positive role in transcript elongation. Although this assumption remains to be established, the observation that transcript elongation in HeLa cells is catalyzed exclusively by RNAP IIO suggests either that the CTD of elongating RNAP II is not a substrate for CTD phosphatase or that dephosphorylation of the CTD causes transcript elongation to cease. The objective of these studies is to gain insights into the regulation of CTD phosphatase activity. Studies presented here indicate that a site on RNAP II that is distinct from the CTD acts as a docking site for CTD phosphatase. Furthermore, TFIIF and TFIIB appear to be involved in the regulation of CTD phosphatase activity.


EXPERIMENTAL PROCEDURES

Materials

Radiolabeled ribonucleotides [-P]ATP (3000 Ci/mmol) and [-P]CTP (800 Ci/mmol) were obtained from DuPont NEN. The Resource Q column was obtained from Pharmacia Biotech Inc. Recombinant mouse CTD was expressed in and purified from Escherichia coli and was kindly provided by Mona Kang (University of California, Davis). The original clone was provided by Bill Dynan (University of Colorado, Boulder). The RAP30 and RAP74 antisera was prepared as described in Wang et al.(16) .

Purification of Proteins

CTD phosphatase was purified from HeLa cells as described by Chambers and Dahmus(9) . RNAPs IIA and IIB were purified from calf thymus by the method of Hodo and Blatti (17) with the modifications described by Kang and Dahmus(13) . RNAP I was purified from calf thymus as described by Dahmus(18) . TFIIB(19) , TBP (20), and TFIIE (21) were expressed in E. coli. TFIIF was expressed in baculovirus and purified as described previously(22) . TFIIH (23) and TFIIJ (24) were purified from HeLa cells. RAP30, RAP74, and RAP74 deletion proteins, each modified with a COOH-terminal His tag, were expressed in E. coli and purified using Ni affinity chromatography.()Although TFIIF, RAP30, and RAP74 form multimeric complexes in solution, for simplicity, the molar amounts are calculated assuming a heterodimeric structure for TFIIF and monomeric structures for RAP30 and RAP74. The DE0.25 transcription extract was purified from HeLa cells as described previously(13) . CTD kinase was purified from the DE0.25 transcription extract. The DE0.25 fraction was loaded on a Resource Q column and developed with a 15-ml linear gradient of 0.1-0.6 M KCl. The peak of CTD kinase activity eluted at 0.23 M and contained CTD kinase 1(25) . Casein kinase II was purified as described previously(26) .

RNAP II Affinity Chromatography

A HeLa cell nuclear extract was chromatographed on either a RNAP II or control column as described previously(27) .

Preparation of P-Labeled Substrates

RNAP IIA was labeled with casein kinase II and [-P]ATP as described by Chesnut et al.(12) . P-Labeled RNAP IIA was incubated with excess cold ATP (2 mM) and CTD kinase to generate RNAP IIO. P-Labeled RNAP IIO was purified by DE52 chromatography as described previously(12) . P-Labeled recombinant CTDo (rCTDo) was prepared by the same procedure using 20 fmol of recombinant CTD except it was not further purified on DE52. P-Labeled RNAP IIO used as a control in experiments with P-labeled CTDo was prepared in the same manner using 20 fmol of RNAP IIA.

P-Labeled subunit IIo was isolated from P-labeled RNAP IIO by electrophoresis on a SDS 5% polyacrylamide gel. The subunit IIo band was located by autoradiography and excised, the protein electroeluted from the gel, the SDS extracted as described previously(28) , and the dried pellet dissolved in and dialyzed against buffer A (50 mM Tris, pH 7.9, 20% glycerol, 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.025% Tween 80, 10 mM KCl, and 10 mM MgCl).

CTD Phosphatase Assay

CTD phosphatase assays were performed in buffer A as described previously(9) . SDS-PAGE analysis of the reactions was carried out according to the method of Laemmli(29) . The resolving gel was either 5% polyacrylamide or a linear gradient of 5-17.5% polyacrylamide. CTD phosphatase activity was quantitated on a BAS1000 PhosphorImager (Fuji) and is given in photo-stimulated luminescence values. One unit of CTD phosphatase converts 1 pmol of RNAP IIO to RNAP IIA in 1 min.

Transcription Reactions

The 822-base pair DNA template containing the adenovirus-2 major late promoter (Ad2-MLP) was prepared essentially as described by Payne et al.(30) . Transcription reactions were performed as described previously (31) with the following modifications. Preinitiation complexes were assembled with 5 µl of DE0.25 (replacing the DE0.15 and DE0.25 fractions), 5-7 10 units of RNAP IIA, 35 ng of the DNA template in a final volume of 31 µl. Reactions were incubated at 30 °C for 30 min. A P-labeled 147-base pair DNA fragment was added to the stop solution to serve as an internal control for recovery of the RNA during the extraction procedure. Transcription reactions with the purified transcription factors were performed as described above except that reactions contained 1.4 pmol of TFIIB, 0.5 pmol of TFIIE, 1.4 pmol of TFIIF, 0.5 µg of TFIIH, 1 µg of TFIIJ, and 1.6 pmol of TBP in place of the DE0.25 fraction.

Fractionation of Preinitiation Complexes with Sepharose CL-4B

Preinitiation complexes were formed in the presence of the DE0.25 extract and P-labeled RNAP IIA in 3.5 transcription reactions as described above. Complexes were fractionated on Sepharose CL-4B as described previously(13) .


RESULTS

CTD Phosphatase Interacts with a Site on RNAP II Distinct from the CTD

Previous studies indicate that CTD phosphatase specifically dephosphorylates phosphoserine and phosphothreonine within the consensus CTD repeat and that it dephosphorylates the CTD in a processive manner(9) . In an effort to further characterize the specificity of CTD phosphatase, its ability to utilize recombinant CTD phosphorylated with CTD kinase was examined. The phosphorylated recombinant CTD is referred to as rCTDo and undergoes the mobility shift in SDS-PAGE characteristic of RNAP subunit IIo (Fig. 1A, compare lanes 1 and 2). Surprisingly, CTD phosphatase did not dephosphorylate rCTDo (Fig. 1A) but did dephosphorylate an equimolar sample of RNAP IIO prepared in the same manner (Fig. 1B). The addition of RNAP IIA did not stimulate dephosphorylation of rCTDo (data not shown). Furthermore, the largest subunit of RNAP IIO, purified by SDS-PAGE, was also not dephosphorylated by CTD phosphatase (Fig. 1C). One interpretation of these results is that a site on native RNAP II outside the CTD must be recognized by CTD phosphatase before it can proceed to dephosphorylate the CTD.


Figure 1: Recombinant CTD and purified subunit IIo are not substrates for CTD phosphatase. The ability of CTD phosphatase to dephosphorylate CTDo, RNAP IIO, and purified subunit IIo was determined as described under ``Experimental Procedures.'' Panel A, aliquots of P-labeled CTDo (0.5 fmol) assayed in the presence of varying amounts of CTD phosphatase and analyzed on a 5-17.5% polyacrylamide-SDS gel. Lane 1, CTDo; lane 2, CTDa; lanes 3-6, 0, 0.1, 0.2, and 0.4 milliunits of CTD phosphatase, respectively, incubated with CTDo. Panel B, aliquots of P-labeled RNAP IIO (0.5 fmol) assayed with CTD phosphatase and analyzed on a 5% polyacrylamide-SDS gel. Lane 1, RNAP IIO; lane 2, RNAP IIO with 0.1 milliunits of CTD phosphatase. Panel C, CTD phosphatase assay with gel-purified P-labeled subunit IIo analyzed on a 5% polyacrylamide-SDS gel. Lane 1, purified subunit IIo; lane 2, subunit IIo incubated with CTD phosphatase; lane 3, mixture of subunit IIo and RNAP IIO incubated with CTD phosphatase.



If indeed, CTD phosphatase interacts with a site on RNAP II prior to dephosphorylation of the CTD, RNAP IIA may act as a competitive inhibitor of CTD phosphatase. To test this idea, CTD phosphatase activity was determined in the presence of increasing amounts of unlabeled RNAP IIA (Fig. 2). The addition of 100 fmol of RNAP IIA to an assay containing 3 fmol of [P]RNAP IIO strongly inhibited CTD phosphatase activity. The addition of comparable amounts of RNAP I or rCTD had no effect on CTD phosphatase activity. However, RNAP IIB, which lacks the CTD, was as effective as RNAP IIA in inhibiting CTD phosphatase activity providing further support for the idea that the recognition site for CTD phosphatase is distinct from the CTD.


Figure 2: RNAP IIA and IIB inhibit CTD phosphatase activity. CTD phosphatase was assayed in the presence of varying amounts of RNAP I, recombinant CTD, RNAP IIA, or RNAP IIB as described under ``Experimental Procedures.'' The CTD phosphatase activity was quantitated on a PhosphorImager and the percent activity remaining plotted versus picomoles of competitor.



CTD Phosphatase Can Inhibit Preinitiation Complex Assembly

If CTD phosphatase stably interacts with a site on RNAP II, high concentrations of phosphatase may interfere with transcription either by preventing the recruitment of RNAP II to the preinitiation complex or by interfering with phosphorylation of the CTD that accompanies transcript initiation. Given that RNAP IIA is in excess in most in vitro transcription reactions, it is unlikely that CTD phosphatase would stimulate the rate of transcription by increasing the amount of RNAP IIA available for recruitment to the promoter. The effect of CTD phosphatase on transcription from the Ad2-MLP was determined as described under ``Experimental Procedures.'' The addition of CTD phosphatase at low concentrations had no effect on transcription in either a crude or reconstituted transcription reaction. However, when the molar ratio of CTD phosphatase relative to RNAP II exceeded 5, transcription was repressed (Fig. 3A). The inhibitory activity co-chromatographs with CTD phosphatase on Mono Q, the last column in the purification of CTD phosphatase (data not shown). The inhibition of transcription is, therefore, likely a direct consequence of CTD phosphatase and not a contaminating activity.


Figure 3: CTD phosphatase inhibits preinitiation complex formation. Panel A, increasing amounts of CTD phosphatase were added to a reconstituted transcription reaction with a fixed amount of RNAP IIA (0.035 pmol) as described under ``Experimental Procedures.'' Lanes 1-4, 0, 0.18, 0.35, and 0.7 pmol of CTD phosphatase added at time 0. Panel B, preinitiation complexes were assembled in the presence of the DE0.25 extract, 0.05 pmol of P-labeled RNAP IIA, and either 1 pmol of CTD phosphatase or in the absence of CTD phosphatase. Reactions were chromatographed on a Sepharose CL-4B column and the fractions analyzed on a 5% polyacrylamide-SDS gel as described under ``Experimental Procedures.'' Panel C, the amount of P-labeled RNAP IIA in each lane was quantitated using a PhosphorImager, the proportion in each lane was calculated relative to the total amount, and plotted versus fraction number. Samples from the reaction with no CTD phosphatase are indicated with filled circles, and samples from the reaction containing CTD phosphatase are indicated with open circles.



Since CTD phosphatase appears to bind directly to RNAP II, the possibility that this interaction interfered with the entry of RNAP II into the preinitiation complex was examined. Preinitiation complexes were formed with P-labeled RNAP IIA in the presence and absence of CTD phosphatase, in the absence of ribonucleotides, and the complexes purified by chromatography on Sepharose CL-4B (Fig. 3B). Preinitiation complexes are excluded from the column and recovered in fractions 10-11 whereas free RNAP II is included and recovered in fractions 13-20. The presence of CTD phosphatase results in a substantial decrease in the assembly of RNAP IIA into preinitiation complexes (Fig. 3, B and C). These results indicate that the association of CTD phosphatase with RNAP II prevents the entry of RNAP II into the preinitiation complex either directly or by preventing RNAP II from binding TFIIF, a factor necessary for the efficient recruitment of RNAP II(32) . Alternatively, CTD phosphatase may bind and sequester a general transcription factor necessary for formation of the preinitiation complex.

The observation that CTD phosphatase inhibits the assembly of RNAP II into a preinitiation complex suggests that it may differentially influence transcription if added prior to or after complex formation. Results presented in Fig. 4A show that CTD phosphatase inhibits transcription if added before preinitiation complex assembly but had a greatly reduced effect if added 1 min after the initiation of transcription (compare lanes 2 and 3 with lanes 5 and 6). Since CTD phosphatase inhibits transcription at a stage before CTD phosphorylation normally occurs, it is unlikely that the inhibitory effect of CTD phosphatase is due to dephosphorylation of the CTD. Furthermore, magnesium is required for CTD phosphatase activity but was not present during the preincubation reaction. Finally, the observation that CTD phosphatase inhibits transcription from both RNAP IIA and IIB establishes that the inhibitory effect is not mediated by the CTD (Fig. 4B).


Figure 4: CTD phosphatase interacts with RNAP II. Panel A, increasing amounts of CTD phosphatase were added to transcription reactions assembled in the presence of the DE0.25 extract and a fixed amount of RNAP IIA (0.025 pmol). CTD phosphatase was added either prior to the formation of a preinitiation complex (lanes 1-3) or after preinitiation complex formation and 1 min after the addition of nucleotides (lanes 4-6). The amount of CTD phosphatase present is indicated at the top of each lane, and the position of the 560 nucleotide runoff transcript is indicated on the left. Panel B, increasing amounts of CTD phosphatase were added to transcription reactions in the presence of the DE0.25 extract and either RNAP IIA (0.025 pmol in lanes 1-3) or RNAP IIB (0.03 pmol in lanes 4-6). CTD phosphatase was added before preinitiation complex formation at a concentration indicated at the top of each lane. Panel C, eluates from an RNAP II (lane 1) and control (lane 2) column were dialyzed against buffer A and assayed for CTD phosphatase activity as described under ``Experimental Procedures.'' The positions of RNAP subunits IIo and IIa are indicated on the left.



To directly test if CTD phosphatase can form a stable complex with RNAP II, a HeLa cell extract was loaded onto an RNAP II affinity column and CTD phosphatase activity determined in salt-eluted fractions. CTD phosphatase activity was detected in the 0.5 M KCl eluate from the RNAP II column but not in the 0.5 M KCl eluate from a control column that did not contain RNAP II (Fig. 4C).

CTD Phosphatase Is Stimulated by TFIIF

In an effort to identify general transcription factors that might influence the activity of CTD phosphatase, highly purified or recombinant general transcription factors were tested individually in a CTD phosphatase assay. The presence of recombinant (baculovirus) TFIIF dramatically stimulates the activity of CTD phosphatase (Fig. 5A). None of the other general transcription factors at the concentrations examined have a stimulatory effect on CTD phosphatase activity. TFIIF does not stimulate the dephosphorylation of RNAP IIO in the absence of CTD phosphatase. The effect of increasing concentrations of TFIIF on CTD phosphatase activity is shown in Fig. 5B. The presence of only 1.3 fmol of TFIIF results in a 2-fold increase in CTD phosphatase activity. Quantitation of the results in this experiment demonstrates that the addition of TFIIF leads to a 3-fold stimulation of activity (Fig. 5C). This is an underestimate due to the assay proceeding past its linear range. In the presence of lower amounts of CTD phosphatase, TFIIF stimulates about 5-fold. Partially purified HeLa cell TFIIF and recombinant TFIIF expressed in E. coli also stimulate CTD phosphatase 5-fold (data not shown). Titration of TFIIF shows that approximately equimolar ratios of TFIIF to RNAP II are sufficient to achieve significant stimulation (Fig. 5C).


Figure 5: TFIIF stimulates CTD phosphatase activity. CTD phosphatase was assayed in the presence of purified transcription factors as described under ``Experimental Procedures.'' Panel A: lane 1, no CTD phosphatase; lanes 2-8, contain CTD phosphatase; lane 2, no transcription factor; lane 3, 1.4 pmol of TFIIB; lane 4, 1.6 pmol of TBP; lane 5, 0.5 pmol of TFIIE; lane 6, 0.4 pmol of TFIIF; lane 7, 0.5 µg of TFIIH; and lane 8, 1 µg of TFIIJ. The amount of the transcription factors chosen was empirically determined as that being optimal for a reconstituted transcription reaction. Panel B, all lanes contain CTD phosphatase; lane 1, no TFIIF; lanes 2-6, 0.5, 1.3, 2.6, 5.2, and 13 fmol of TFIIF, respectively. Panel C, the CTD phosphatase activity from panel B was quantitated on a PhosphorImager and the photo-stimulated luminescence values plotted versus the TFIIF/RNAP IIO molar ratio. The assay contained 3 fmol of RNAP IIO. For simplicity, moles of TFIIF were calculated assuming a dimeric structure.



Since CTD phosphatase activity is observed in the absence of exogenous TFIIF, TFIIF appears to stimulate a basal level of CTD phosphatase activity. Alternatively, TFIIF may be essential for CTD phosphatase activity and be present in suboptimal levels in the CTD phosphatase and/or RNAP II preparations. To test this, aliquots of CTD phosphatase, RNAP IIA, and the CTD kinase used in the preparation of RNAP IIO were analyzed on an immunoblot using an antisera raised against TFIIF. No TFIIF was detected in the CTD phosphatase, RNAP IIA, or the CTD kinase preparations even though the assay was capable of detecting less than 1 ng (data not shown). Therefore, reactions assembled in the absence of exogenous TFIIF must contain less than 0.1 fmol of TFIIF. This result indicates that TFIIF is not essential for a basal level of CTD phosphatase activity.

The COOH-terminal Domain of RAP74 Is Sufficient to Stimulate CTD Phosphatase

TFIIF is composed of two subunits, RAP30 and RAP74. The ability of individual subunits to influence CTD phosphatase activity was determined. The RAP74 subunit stimulates CTD phosphatase activity whereas the RAP30 subunit has no effect (Fig. 6). Furthermore, RAP30 has no effect on the activity of RAP74 or TFIIF, even if added in a 200-fold molar excess prior to the addition of RAP74 or TFIIF (data not shown). RAP74 stimulates CTD phosphatase activity more than 20-fold in contrast to TFIIF which stimulates only 5-fold. However, TFIIF is more stimulatory at low concentrations than is RAP74 (data not shown). For full stimulation by TFIIF, approximately 20 fmol is required, whereas 500 fmol of RAP74 still does not show saturation.


Figure 6: The RAP74 subunit of TFIIF stimulates CTD phosphatase activity. CTD phosphatase was assayed in the presence of TFIIF, RAP30, and RAP74 as described under ``Experimental Procedures.'' All lanes contain CTD phosphatase. Lane 1, CTD phosphatase alone; lane 2, TFIIF (0.2 pmol); lane 3, RAP30 subunit (3 pmol); lane 4, RAP74 subunit (4.3 pmol).



The sequence of RAP74 suggests it is organized into a NH- and COOH-terminal domain and a highly charged central domain(33) . A panel of RAP74 proteins deleted in various regions was tested to locate the region in RAP74 responsible for stimulating CTD phosphatase (Fig. 7). CTD phosphatase activity was determined in the presence of 500 fmol of each of the RAP74 deletion proteins. Like RAP74, this concentration does not saturate the assay. The smallest protein able to stimulate CTD phosphatase contains residues 358-517 and stimulates about 5-fold. All active deletion proteins contain this region, and proteins from which this region is deleted (1-409, 407-517) are inactive. However, two of the deletion proteins that contain residues 358-517 are inactive (205-517, 136-258), both of which contain portions of the central charged region. Deletion of the central charged region (137-356) results in a fully active molecule. Construct 137-356 is even more active than the wild type RAP74 at lower concentrations resulting in 20-fold stimulation in the presence of only 50 fmol (data not shown). The NH-terminal region of RAP74 is also important for CTD phosphatase stimulatory activity. Deletion of residues 1-74 has little effect, whereas deletion of a further 13 residues, encompassing a highly conserved region, results in a dramatic loss of activity (Fig. 7, compare lanes 15 and 16). Combination of two inactive fragments encompassing the entire subunit of RAP74 (1-409 and 407-517, 1-205 and 207-517) failed to reconstitute CTD phosphatase stimulatory activity (data not shown).


Figure 7: Stimulatory activity of RAP74 deletion constructs. CTD phosphatase was assayed in the presence of 500 fmol of RAP74 deletion proteins as described under ``Experimental Procedures.'' Lane 1, no CTD phosphatase; lane 2, CTD phosphatase alone; lane 3, CTD phosphatase and TFIIF; lane 4, CTD phosphatase and RAP74; lanes 5-20, CTD phosphatase and deletion proteins as designated in the left margin. The numbers on the left refer to the amino acid residues present except where the numbers are preceded by a , in which case the numbers reflect the deleted region. CTD phosphatase activity was quantitated on a PhosphorImager and the fold stimulation calculated relative to CTD phosphatase alone. Due to the small linear range of the assay, stimulatory values for 1-517, 74-517, and 137-356 were measured in independent experiments with the CTD phosphatase titrated into the linear range.



TFIIB Inhibits the Ability of TFIIF to Stimulate CTD Phosphatase

To further investigate the effect of the general transcription factors on CTD phosphatase activity, each general transcription factor was added to assays containing TFIIF and CTD phosphatase. TFIIB, and to a lesser extent TBP, inhibits the TFIIF stimulated activity (Fig. 8, A and B). TFIIB had no effect on CTD phosphatase activity in the absence of TFIIF (Fig. 8B, compare lanes 2 and 3). In the presence of TFIIF, TFIIB inhibits CTD phosphatase activity to basal levels (Fig. 8B, compare lanes 2 and 3 with 8). TFIIB also inhibits the RAP74 stimulated assay (Fig. 8A, compare lanes 7 and 8). High amounts of TBP (1600 fmol) partially inhibit CTD phosphatase activity in the presence or absence of TFIIF. The inhibitory effect of TBP may be a consequence of its binding to the CTD (34). Little effect is seen with less than 500 fmol of TBP. The presence of 30 fmol of TFIIB in an assay containing 100 fmol of TFIIF results in a 75% reduction in the stimulatory activity of TFIIF (Fig. 8C).


Figure 8: TFIIB inhibits the stimulatory effect of TFIIF. Panel A, CTD phosphatase and 50 fmol of TFIIF or 500 fmol of RAP74 were assayed in the presence of the general transcription factors as described under ``Experimental Procedures.'' Lane 1, CTD phosphatase and TFIIF alone; lane 2, plus 1.4 pmol of TFIIB; lane 3, plus 1.6 pmol of TBP; lane 4, plus 0.5 pmol of TFIIE; lane 5, plus 0.5 µg of TFIIH; lane 6, plus 1 µg of TFIIJ; lane 7, CTD phosphatase and RAP74 alone; and lane 8, plus 270 fmol of TFIIB. Panel B: lane 1, no CTD phosphatase; lane 2, CTD phosphatase; lane 3, CTD phosphatase and 270 fmol of TFIIB; lanes 4-8, contain 0, 8, 27, 80, and 270 fmol of TFIIB, respectively, 100 fmol of TFIIF and have half as much CTD phosphatase as lanes 2 and 3. Panel C, the CTD phosphatase activity from lanes 4 to 8 of panel B was quantitated on a PhosphorImager, the fold stimulation calculated, and plotted versus fmol of TFIIB. Assays contained 3 fmol of RNAP IIO.




DISCUSSION

The following results support the idea that there is a docking site on RNAP II to which CTD phosphatase binds and thereby gains access to the CTD. First, RNAPs IIA and IIB are competitive inhibitors of CTD phosphatase activity. The docking site must be outside the CTD since RNAP IIB, which lacks the CTD, is as effective as RNAP IIA in inhibiting CTD phosphatase activity. Second, RNAP II can form a stable complex with CTD phosphatase as indicated by the observations that CTD phosphatase (a) binds to a RNAP II affinity column and (b) prevents the assembly of RNAP IIA into the preinitiation complex. Third, rCTDo cannot be dephosphorylated by CTD phosphatase. An alternative interpretation of the finding that rCTDo is not a substrate is that the conformation of the phosphorylated CTD in RNAP IIO is different from that of the recombinant CTD and that CTD phosphatase recognizes only the native conformation. This is unlikely given the apparent independence of the CTD from the core of RNAP II and that CTD kinases in general phosphorylate both rCTD and RNAP IIA. Furthermore, this interpretation does not account for the fact that RNAPs IIA and IIB are competitive inhibitors of the phosphatase reaction.

The indication that CTD phosphatase must first interact with a site on RNAP II distinct from the CTD, before the CTD can be dephosphorylated, has important implications for the regulation of CTD phosphatase activity. The accessibility of the docking site could control the entry of CTD phosphatase and hence dephosphorylation of the CTD. The accessibility of the docking site may be influenced by the association of transcription factors and/or the conformation of RNAP II in the initiation, elongation, and termination complex. This is in contrast to a model in which CTD phosphatase has ready access to the extended CTD.

Many protein phosphatases contain targeting subunits that bind at specific sites to localize the phosphatase to its site of action(35) . The domain in CTD phosphatase that binds to the docking site on RNAP II may be analogous to a targeting subunit and would explain the specificity of CTD phosphatase for RNAP II. The process of docking could account for the processive behavior of CTD phosphatase and may also be required to generate an active conformation of CTD phosphatase (Fig. 9). Alternatively, the docking process may ``load'' CTD phosphatase onto the CTD which could then disengage from the docking site and proceed along the CTD to dephosphorylate it.


Figure 9: Model of CTD phosphatase action. CTD phosphatase is represented by the white oval, RNAP II by the shaded area, and CTD by the wavy line. Details of the model are described in the text.



The results presented above indicate that TFIIF may be involved in the regulation of CTD phosphatase activity. TFIIF is a heterotetramer of RAP30 and RAP74 and both subunits can interact with RNAP II directly (22). During initiation, TFIIF suppresses the non-selective binding of RNAP II to DNA, facilitates the recruitment of RNAP II to the preinitiation complex, and stabilizes the preinitiation complex(22) . In addition, TFIIF stimulates elongation by reducing pausing of RNAP II (36). Results presented above show that TFIIF is also capable of stimulating the dephosphorylation of RNAP IIO by CTD phosphatase. The mechanism by which TFIIF stimulates CTD phosphatase is not known but could affect any of several steps, such as the binding of CTD phosphatase to RNAP II or the subsequent dephosphorylation reaction.

The stimulatory activity of TFIIF appears to reside in the RAP74 subunit. The addition of RAP30, either alone or in combination with RAP74, has no effect on CTD phosphatase activity. However, since it is unlikely that RAP30 and RAP74 assemble into intact TFIIF under these conditions, the possibility that RAP30 participates directly in TFIIF mediated stimulation cannot be excluded. Interestingly, TFIIF and RAP74 differ in their level of stimulation, 5- and 20-fold, respectively, and the amount required for stimulation. The ability of TFIIF to stimulate CTD phosphatase at lower concentrations than RAP74 suggests that TFIIF has a higher affinity for RNAP II than the RAP74 subunit.

The minimal region of RAP74 sufficient for stimulation is the COOH-terminal region spanning residues 358-517. This COOH-terminal region includes an RNAP II binding site and accessibility of this site is affected by NH-terminal and central regions of RAP74.()Central sequences of RAP74 mask RNAP II binding while NH-terminal sequences counteract this effect. This COOH-terminal region of RAP74 also stimulates accurate transcription in vitro although it is not necessary for initiation(36, 38) . The presence of the NH-terminal region increases CTD phosphatase stimulation from 5- to 20-fold and is absolutely required for activity if the central region is present. This observation is consistent with the effect of NH-terminal sequences in reducing the masking effect of central sequences on RNAP II binding. Particularly critical are residues 74-87 that encompasses a highly conserved region containing mostly charged residues. Interestingly, the region involved in binding RAP30 (residues 1-172) spans the region which is critical for the 20-fold stimulation and may explain the different levels of stimulation by TFIIF and RAP74.

TFIIB can bind to both RNAP II and TFIIF and was found to inhibit TFIIF-stimulated CTD phosphatase activity. It is important to note that TFIIB does not influence the basal level of CTD phosphatase activity. TFIIB functions early in initiation and facilitates the recruitment of RNAP II/TFIIF. The mechanism by which TFIIB facilitates the recruitment of RNAP II is not clear but likely involves a direct interaction of TFIIB with RNAP II, RAP30, and/or RAP74(37, 39) .

The finding that CTD phosphatase gains access to the CTD by interaction with a docking site on RNAP II distinct from the CTD and that both TFIIF and TFIIB influence CTD phosphatase activity suggests that the regulation of CTD dephosphorylation may be complex. One interpretation of these results is that the association of TFIIF (or RAP74) with RNAP II facilitates the interaction of CTD phosphatase with RNAP II either by a direct interaction between TFIIF and CTD phosphatase or by inducing a conformational change in RNAP II that makes the docking site more accessible. CTD phosphatase activity would then be stimulated at specific steps in the transcription cycle by the interaction of TFIIF with RNAP II.

TFIIF is a component of the preinitiation complex and could in principle promote dephosphorylation of the CTD during initiation. However, phosphorylation of RNAP II occurs during the transition to a stable elongation complex, and although its role in this transition remains to be defined, the stimulation of dephosphorylation of RNAP II at this stage appears antagonistic to the transcription process. TFIIB may function at this step to inhibit the normal stimulation of CTD phosphatase activity by TFIIF. In addition, the binding site for CTD phosphatase may be occluded in the preinitiation complex. To test these ideas, it will be of considerable interest to examine the ability of CTD phosphatase to dephosphorylate RNAP II at various stages in the transcription cycle.


FOOTNOTES

*
This research was supported by Grants GM40708 (to Z. F. B.) and GM33300 (to M. E. D.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 916-752-3551; Fax: 916-752-3085.

The abbreviations used are: RNAP, RNA polymerase; Ad2-MLP, adenovirus-2 major late promoter; CKII, casein kinase II; CTD, COOH-terminal domain; PAGE, polyacrylamide gel electrophoresis; RAP, RNAP associating protein; TBP, TATA-binding protein; TF, transcription factor.

R. S. Chambers and M. E. Dahmus, unpublished observation.

B. Q. Wang and Z. F. Burton, submitted for publication.


ACKNOWLEDGEMENTS

We gratefully acknowledge Jon Chesnut for preparation of the general transcription factors, Grace Dahmus for preparation of RNAP I, RNAP IIA, and CKII and technical assistance, and Mona Kang for preparation of RNAP IIB and recombinant CTD. We also thank Mona Kang, Sang Soo Lee, and Alan Lehman for their helpful comments during the course of these studies and for their review of this manuscript.


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