Proc. Natl. Acad. Sci. USAVol. 88, pp. 10004-10008, November 1991Biochemistry

The nonphosphorylated form of RNA polymerase II preferentiallyassociates with the preinitiation complexHUA Lu, OSVALDO FLORES, ROBERTO WEINMANN, AND DANNY REINBERG*Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854

Communicated by Mark Ptashne, July 22, 1991 (received for review April 1, 1991)

ABSTRACT The two forms of RNA polymerase H thatexist in vivo, phosphorylated (HO) and nonphosphorylated(HA), were purified to apparent homogeneity from HeLa cells.The nonphosphorylated form preferentially binds to the pre-initiation complex. RNA polymerase H in the complex wasconverted by a cellular protein kinase to the phosphorylatedform.

Purified RNA polymerase II cannot accurately initiate tran-scription from class II promoters in vitro unless it is supple-mented with general transcription initiation factors (1-3).Seven human general transcription factors (TFIIA, -IIB,-IID, -lIE, -IIF, -IIG, and -IIH) that, together with RNApolymerase II are sufficient for specific transcription, havebeen identified (O.F. and D.R., unpublished results).

Despite progress in the purification of the general tran-scription factors, determination of their individual activitiesand contributions to the formation of a transcription-competent complex remains obscure. Complicating theseanalyses is the existence in eukaryotic cells of two differentforms ofRNA polymerase II, IIA and IIO, which differ in thelevel of phosphorylation of a highly conserved heptapeptiderepeat present at the carboxyl terminus of the largest subunit(carboxyl-terminal domain; CTD) (4-6). The heptapeptiderepeat is essential for viability (7-10); nevertheless, a thirdspecies ofRNA polymerase II lacking the CTD (IIB) has beenobserved in vitro (11-14). Photoaffinity labeling experimentsdemonstrated that nascent RNA transcripts crosslink almostexclusively to the phosphorylated IIO form in vivo and invitro (5, 15, 16), suggesting that it is the IIO polymerase thatelongates RNA chains. Monoclonal antibodies against thenonphosphorylated IIA form inhibited specific transcriptioninitiation (17, 18), suggesting that RNA polymerase IIA wasmore active than IIO during specific transcription initiation invitro. Therefore, the CTD phosphorylation state may regu-late the transition from initiation to elongation (19).We have purified human RNA polymerases IIO and IIA to

apparent homogeneity and analyzed their roles in transcrip-tion. We demonstrate that the IIA form associates preferen-tially with the preinitiation complex where it is then con-verted by a cellular protein kinase to the phosphorylated 11Oform.

MATERIALS AND METHODSTranscription Factors, Transcription Reaction Mixtures,

and DNA Binding Assays. Transcription reactions and DNAbinding assays were performed as described (20). Transcrip-tion factor IIA (TFIIA) (P. Cortes and D.R., unpublisheddata), TFIIB (22), TFIIE (23), and TFIIF (24) were purifiedfrom HeLa cell nuclear extracts as described. TFIID waspurified to homogeneity as described (20) from recombinantEscherichia coli cells expressing the yeast TFIID gene (25).

Purification of the Phosphorylated (HO) and Nonphospho-rylated (HA) Forms of RNA Polymerase H. RNA polymeraseII was purified from HeLa cell nuclear pellets (7.5 x 1010cells). Enzyme solubilization and chromatography on DE-52were as described (26). Active fractions ofthe DE-52 column,between 0.2 and 0.3 M salt, were pooled (109 mg of protein,170 ml), diluted to 0.2M ammonium sulfate (the same salt wasused in the following steps) with buffer D [50 mM Tris HC1,pH 7.9/25% (vol/vol) glycerol/0.1 mM EDTA/2 mM dithio-threitol/0.2 mM phenylmethylsulfonyl fluoride/pepstatin Aat 25 ug/ml], and centrifuged at 20,000 rpm for 20 min in aSorvall T647.5 rotor. The supernatant was loaded onto aheparin-agarose column (5 mg of protein per ml of resin)previously equilibrated with buffer D containing 0.2 M salt.The RNA polymerase II activity was eluted with a lineargradient (five column volumes) from 0.2 to 0.7M salt in bufferD. The active fractions (at 0.3-0.4 M salt) were pooled (15 mgof protein, 38 ml), and the ionic strength was adjusted to 0.9M with 3 M ammonium sulfate. Samples were then centri-fuged at 20,000 rpm for 20 min (Sorvall TFT50.38 rotor), andthe supernatant was loaded onto an HPLC TSK-phenylcolumn (75 x 7.5 mm) equilibrated with buffer B (identical tobuffer D except that the glycerol concentration was 20%)containing 0.9M salt. RNA polymerase II activity was elutedwith a linear gradient from 0.9 to 0.0 M salt in buffer B. Theactive fractions (at around 0.3 M) were pooled (2.1 mg ofprotein, 7 ml) and directly loaded onto an HPLC DEAE-5PW column (8 x 7.5 mm) equilibrated with buffer B con-taining 0.2 M salt. RNA polymerase II was eluted with alinear gradient from 0.2 to 0.7 M salt in buffer B. The activitywas eluted at about 0.4 M salt.

Fractionation of the enzyme (mixture of IIO and IIA) onthe HPLC TSK-phenyl column resulted in the irreversibleretention of the IIA form (data not shown). Thus, in order toisolate the IIA form, fractionation on the TSK-phenyl columnwas omitted, and the heparin-agarose pool (22 mg in 45 ml)was directly loaded onto an HPLC DEAE-5PW column.The activity was eluted as described above. The IIO form waseluted at -0.4 M salt, whereas the IIA form was eluted atabout 0.5 M salt. The active fractions containing the IIA formwere pooled (2.4 mg, 10 ml). The salt concentration wasadjusted to 1.3 M, and the pool was loaded onto an FPLCalkyl-agarose column (80 x 7.5 mm) equilibrated with bufferB containing 1.3 M salt. The activity was eluted with a lineargradient of ammonium sulfate (1.3 M-0.0 M) in buffer B. Theactive fractions (eluting at -0.9 M salt, 0.3 mg in 4 ml) werepooled and dialyzed against buffer B containing 0.05 Mammonium sulfate. RNA polymerase IIA was further puri-

Abbreviations: CTD, carboxyl-terminal domain; TF, transcriptionfactor; Ad-MLP, adenovirus major late promoter; DAB, DNA-protein complex formed at the TATA motif of the Ad-MLP thatincludes TFIIA, -lID, and -IIB; CIP, calf intestine phosphatase;BSA, bovine serum albumin; DABPolF, DNA-protein complex thatincludes the DAB complex, RNA polymerase II, and TFIIF.*To whom reprint requests should be addressed at: Department ofBiochemistry, University of Medicine and Dentistry ofNew Jersey,675 Hoes Lane, Piscataway, NJ 08854-5635.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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fied by chromatography on an FPLC Mono Q column equil-ibrated with buffer B containing 0.05 M ammonium sulfate.The column was first washed with buffer B containing 0.15 Msalt, and the activity was eluted with a 0.35 M wash. Activefractions were stored at -80'C.The amount of protein in the final step of purification of

RNA polymerase II was determined by using the Lowryreagent and also by a quantitative Western blot analysis usingantibodies against exon 5 of the largest subunit of RNApolymerase II (27).

Protein Kinases Phosphorylating the CTD of the LargestSubunit of RNA Polymerase II. Two different protein kinasesthat phosphorylate the CTD were detected and purified fromHeLa cell extracts. The assay employed was as described(28) and utilized RNA polymerase IIA as a substrate. One ofthe kinases (Hkl) copurified with TFIIH up to the DEAE-5PW protein fraction (O.F. and D.R., unpublished results).Hkl was separated from TFIIH by chromatography on aMono S column. TFIIH eluted at about 0.35 M KCI, whereasHkl activity eluted at 0.23 M KCI. Hk2 activity has beenextensively purified from the TFIID protein fraction andidentified as maturating promoter factor (MPF) (H.L. andD.R., unpublished results).Recovery of RNA Polymerase from DNA-Protein Com-

plexes. DNA-protein complexes were formed on the adeno-virus major late promoter (Ad-MLP) and analyzed as de-scribed (20). Complexes were visualized by exposing the wetgels to Kodak XR-5 film. Complexes were excised, andproteins were eluted at 370C by using buffer X (50 mMNH4HCO3, pH 8.0/0.2 mg of lysozyme per ml/0.2 mMEDTA/0.1% SDS). The eluted proteins were dried andwashed with cold trichloroacetic acid (20%). Proteins wereresuspended in loading buffer (9 M urea/1% Nonidet P-40/62.5 mM Tris-HCl, pH 6.8/5% 2-mercaptoethanol) and sep-arated by SDS/PAGE. Proteins were transferred to nitrocel-lulose membranes by Western blotting. The membranes wereincubated with first antibody and then with enhancedchemiluminescence detection reagents I and II as describedby the manufacturer (Amersham).

A

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FIG. 1. Purification of the 110 and IIA forms ofRNA polymeraseII. (A) An aliquot (15 ,ul) of purified RNA polymerase 110 was loadedon an SDS/5-17% PAGE gel, and the proteins were visualized bysilver staining. Migration ofprotein standards (in kDa) is indicated onthe left. Migration of individual RNA polymerase II subunits (in kDa)is indicated at the right. (B) Silver staining of an SDS/5-17% PAGEgradient gel containing 15 ,ul of purified RNA polymerase IIA, 40 ,ulof purified TFIIE, and 30 ,ul of purified TFIIB. Subunit compositionofRNA polymerase II is indicated on the left. Molecular size markers(in kDa), TFIIB, and the subunit composition of TFIIE are indicatedon the right. (C) Western blot analysis of the largest subunit ofRNApolymerase II present in crude (lane 3), purified 110 (lane 1), purifiedIIA (lane 4), and phosphatase-treated (CIP) 11O (lane 2) preparations.The antibodies used were against a,-galactosidase fusion proteincontaining sequence present in exon 5 of the largest subunit ofRNApolymerase 11 (27). The arrows on the left indicate the phosphoryl-ated (Iho) and nonphosphorylated (Ila) largest subunits of RNApolymerase II. The broken arrow indicates the migration of theCIP-treated 110 form of the enzyme.

RESULTSPurification of the IIO and HA Forms ofRNA Polymerase H.

To determine whether the 110 and IIA forms of RNApolymerase II play different roles during the transcriptioncycle, we purified each form of the enzyme to apparenthomogeneity as described in Materials and Methods. Thereare three forms of the largest subunit in most preparations ofRNA polymerase II thus far described: IIO (phosphorylated,240 kDa), IIA (nonphosphorylated, 200 kDa), and IIB (pro-teolyzed, 170 kDa) (12). When purified RNA polymerase IIOwas analyzed on an SDS/PAGE gel and visualized by silverstaining, 10 polypeptides could be distinguished (Fig. 1A),but only the 240-kDa, Ilo form of the largest subunit waspresent. The polypeptide composition of the IIA enzyme isshown in Fig. 1B. It was also composed of 10 differentpolypeptides that are known to constitute the core enzyme(10, 12, 29), but only the Ila form of the largest subunit waspresent.To substantiate the homogeneity of the IIO and IIA spe-

cies, the enzyme preparations were analyzed by Western blotusing antibodies against sequences present in exon 5 of thelargest subunit of RNA polymerase II, which recognize withsimilar affinities both the 110 and IIA forms of the enzyme(27). When a crude RNA polymerase II protein preparationwas used in the Western blot analysis, the antibodies reactedwith two bands with apparent molecular masses of -240 and200 kDa (Fig. 1C, lane 3). Analysis ofthe separated 110 or IIAenzyme preparations showed that the antibodies reacted onlywith bands of 240 kDa or 200 kDa, respectively (Fig. 1C,

lanes 1 and 4). In addition, treatment of the 11O preparationwith calf intestine phosphatase converted the 240-kDa poly-peptide to multiple polypeptides migrating between the 240-kDa and 200-kDa polypeptides (Fig. 1C, lane 2). Thus, theprocedure described resulted in RNA polymerase II prepa-rations containing either the nonphosphorylated (IIA) orphosphorylated (IIO) form of the enzyme.The Nonphosphorylated Form of RNA Polymerase II Pref-

erentially Binds to the Preinitiation Complex. Entry of RNApolymerase II into the transcription cycle was demonstratedto require a DNA-protein complex formed at the TATA motifof the Ad-MLP that included TFIIA, -IID, and -IIB (DABcomplex) (30, 31). The binding of RNA polymerase II to theDAB complex was dependent on TFIIF (30). Using highlypurified RNA polymerase II and transcription factors, weanalyzed whether both forms of the enzyme could associatewith the preinitiation complex. A DNA-protein complex thatincludes the DAB complex, RNA polymerase II, and TFIIF(DABPolF) was formed in each case, but with differentmobilities (Fig. 2A, see lanes 9-17). Approximately 80% oftheDAB complex was shifted to the DABPolF complex when40 ng of IIA enzyme was added to the assay (Fig. 2A, lane 9);however, no more than 30%o of the DAB complex was shiftedto the DABPolF complex when an equal amount of RNApolymerase 110 (40 ng) was used (Fig. 2A, lane 17). CompleteDAB to DABPolF conversion required an -4-fold increase inthe amount of11O polymerase (140 ng; see Figs. 3 and 4). Thereduced ability of RNA polymerase 110 to recognize theDAB complex was not due to inactivity of the enzyme

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A ~~~~~D+A+B+F+Pol

+10o201304060-- - -I0'2030:4& no (n g+ +:40 3&2010-10-200 lla1ng)

DAB Po lEo FDABP~lU0FDAPl -F-

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performed as described (20). Lanes 1 and 2 represent reactionproducts formed with TFIIA and TFIID and with TFIIA, TFIID, andTFIIB. Lanes 3-17 contained various amounts of RNA polymerase11A and/or polymerase 110 as indicated. At the left, migration ofcomplexes is indicated: DA and DAB (short lines) and also DABPoIFformed with the 11O (solid arrow) and IIA (bracket) forms of thepolymerase. The dots beside lanes 5, 6, and 8 indicate the migrationof the bands characteristic of the different DABPolF complexes.Bands representing the different DNA-protein complexes were cutout of the gel and assayed in a liquid scintillation counter. (B) Theproteins present in the DABPolF DNA-protein complex formed witheither the IIA (lanes 2 and 3) or 110 (lanes 4 and 5) polymerase wereextracted from the complex and separated by electrophoresis on an

SDS/5% PAGE gel. The form of the enzyme in the complex was

analyzed by Western blotting using the enhanced chemilumines-cence technique and antibodies directed against exon 5 of the largestsubunit of RNA polymerase 11 (27). In this experiment the proteinsloaded in lanes 2-5 were purified from complexes derived from 10individual binding reactions (40 ng of RNA polymerase II perreaction). Lanes 3 and 5 represent the products of DNA bindingreactions performed in the presence of ATP (1 mM). Lanes 1 and 6contained 20 ng of RNA polymerase IIA or IIO, as indicated.Migration of the IIO, IIA, and IIB subunits of RNA polymerase isindicated. (C) DABPolF DNA-protein complexes were derived frombinding reactions containing RNA polymerase IIA and/or polymer-ase 110 in the amounts and combinations indicated at the top of lanes1-3. DABPolF (lanes 1-3) and DAB (lane 4) complexes (derived fromfive different reactions) were excised from the gel, and subunits ofthe enzyme were separated by electrophoresis on an SDS/5-17%PAGE gradient gel and analyzed by Western blotting as described inB. Lanes labeled Ho and Ila represent lanes in which RNA poly-merase 11O and IIA were directly loaded onto the gel. The migrationof the 11O and IIA subunits is indicated at the left.

because polymerase activity measured in a promoter-independent transcription assay showed the 11O form to bemore active (data not shown). Also, both preparations ofRNA polymerase II had similar DNA binding activity as

measured by using single-stranded DNA (data not shown).To analyze whether the differences observed were due to

different affinities of the IHO and IIA enzyme, complexformation was analyzed by using constant amounts (40 ng) ofpolymerase but various ratios of polymerase IIA to polymer-ase IIO. By exploiting the mobility differences between theDABPolIIAF and DABPolIIOF complexes, it was possible to

compare the amount of each complex formed. When equalamounts (20 ng) of 110 and IIA polymerase were added to theDNA binding assay, the DNA-protein complex formed mi-grated almost identically to that formed with RNA polymer-ase IIA alone (Fig. 2A, lane 5).To directly analyze the form of RNA polymerase II that

was associated with the DABPolF DNA-protein complex,the proteins present in the complexes were recovered andanalyzed by Western blot using antibodies directed againstthe largest subunit of RNA polymerase II (Fig. 2B). Theseantibodies recognize with about the same affinity both the11O (Fig. 2B, lane 6) and IIA (Fig. 2B, lane 1) forms of theenzyme (27). The largest subunit of RNA polymerase IIextracted from DNA-protein complexes formed with IIA orIHO polymerase comigrated with the IIA and 11O forms of theenzyme, respectively (Fig. 2B, compare lanes 2 and 3 withlanes 4 and 5). Thus both forms of RNA polymerase II canassociate with the DABPolF DNA-protein complex, and noconversion from one form to the other occurs in the DAB-PoIF complex, independently of whether the reactions wereperformed in the presence or absence of ATP (Fig. 2B,compare lanes 2 and 4 with 3 and 5, respectively). Consistentwith the results presented in Fig. 2A, the amount of RNApolymerase II recovered from complexes formed with the 110polymerase was lower than that recovered from complexesformed with the IIA polymerase, even though equal amountsof enzyme (40 ng) were used. When the DABPolF DNA-protein complex was formed with equal amounts of RNApolymerase IIA and 11O (20 ng), RNA polymerase IIA waspreferentially recovered from the complex (Fig. 2C, lane 3).An almost equal amount of 11O and IIA enzyme could berecovered from the DABPolF complex only when the inputratio of IHO to IIA enzyme was 3:1 (Fig. 2C, lane 2).To analyze whether the results obtained above were due to

different affinities and/or stabilities of the 11O and IIA en-zymes in the DABPolF complex, the rate of association ofeach form ofthe enzyme with the DAB complex was analyzed.A constant amount ofTFIIF and RNA polymerase 110 or IIAwas added to DNA binding assay mixtures containing theDAB complex, and aliquots were removed at different times.After 1 min of incubation o90% of the DAB complex waspresent as DABPolF complex in reactions containing the IIAenzyme; however, even after 20 min ofincubation only 60% ofthe DAB complex was complexed as DABPolF in reactionscontaining the 11O enzyme (Fig. 3A). The rate of associationof the 110 polymerase with the DAB complex could not beaugmented by increasing the concentration ofRNA polymer-ase II. We found that the extent ofthe reaction, but not the rateof association, was dependent on enzyme concentration;increasing the concentration of polymerase 11O by 3-fold (to180 ng) resulted in a slow but complete conversion ofthe DABto the DABPolF complex; on the other hand, decreasing theconcentration of polymerase IIA by half (to 30 ng) resulted in-60%o conversion of the DAB to the DABPolF complex,without affecting the reaction rate (data not shown).The results presented above strongly suggest that RNA

polymerase IIA preferentially associates with the DAB com-plex. If this were true, one would expect that conversion ofthe IHO form of polymerase to the IIA, by phosphatasetreatment in vitro, should render a polymerase that associatesmore effectively with the preinitiation complex. Equalamounts ofRNA polymerase 110 were incubated either withcalf intestine phosphatase (CIP) or bovine serum albumin(BSA). Aliquots were then withdrawn and assayed for theirability to form the DABPolF complex. The addition of80 and160 ng of CIP-treated 11O polymerase resulted in -70% and100% conversion of the DAB to the DABFPol complex,respectively (Fig. 3B, lanes 7 and 8). BSA-treated ITO poly-merase was less effective in producing the DABPolF complexbecause 160 ng of polymerase converted only 50%o of the

B

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Phosphorylation ofRNA Polymerase HA in the PreinitiationComplex by a Protein Kinase. In light of the studies presentedabove, we investigated whether RNA polymerase IIA couldbe converted to the phosphorylated form by a protein kinase.In contrast to studies of others (17, 19), we observed thatnone of the general transcription factors contained suchactivity (data not shown). However, a protein kinase capableof phosphorylating the IIA polymerase was isolated fromHeLa cell nuclear extracts. This protein kinase (Hkl; seeMaterials and Methods), in the presence of ATP, convertedthe DABPolIIAF complex (Fig. 4A, lane 4) to one thatcomigrated with the DABPolIIOF complex (Fig. 4A, com-pare lane 5 with lane 3). The change in migration of thecomplex was due to phosphorylation ofRNA polymerase IIAbecause the enzyme recovered from the shifted complexcomigrated on a SDS/PAGE gel with the IIO form isolatedfrom HeLa cells (Fig. 4C, compare lanes 4 and 2). Theconversion required ATP or dATP or GTP (Fig. 4B) and anRNA polymerase II in association with the preinitiation

-Ir. complex. Separately, the protein kinase, in the presence of- Ia [Y-32P]ATP, labeled the IIA form but did not convert it to the

IIO form (data not shown). Similar results were observedwith MPF (data not shown).

DISCUSSIONTwo forms of RNA polymerase II exist in vivo (4-6), whichdiffer by phosphorylation of an unusual heptamer repeat

A

1 2 3 4 5 6 7 8

FIG. 3. Kinetics of association ofRNA polymerase IIA and 11Owith the preinitiation complex. (A) DNA binding reactions wereperformed as described in Fig. 2. During the first incubation complexDAB was formed on the Ad-MLP. After 60 min, RNA polymerase11 (60 ng), as indicated, together with TFIIF, was added to thereaction mixtures, which were then incubated for different times, asindicated in the figure. Complexes were separated by electrophoresison a native polyacrylamide gel, and the dry gel was exposed to XR-5film. Bands were cut of the gel and assayed in a liquid scintillationcounter. The percent ofDAB and DABPolF complexes are presentedin the graph. DAB(IIa) and DAB(IIo) denote complexes remainingafter addition of the IIA or 110 forms of RNA polymerase II,respectively. (B) RNA polymerase 110 (90 ,g/ml) was incubatedwith CIP (2 jtg) or BSA (2 .&g) for 60 min at 250C as described (32).Different amounts of treated RNA polymerase (as indicated abovelanes 4-8) were added to DNA binding assays. Mixtures wereincubated for 30 min, and the products separated by electrophoresison a native polyacrylamide gel. Lanes 1-3, reactions performed withuntreated samples. (C) Different forms of RNA polymerase 11 (110,IIA, and 110 treated with CIP, as indicated) were separated byelectrophoresis on an SDS/5% PAGE gel and visualized by silverstaining. Migration of the largest and second largest subunits (IG)are indicated on the right. A 200-kDa molecular size marker (myosinH chain) is shown on the left.

DAB complex and full conversion to the DABPolF complexrequired -320 ng of enzyme (Fig. 3B, lanes 4-6). Theconcentration of RNA polymerase II used in this assay washigher than in the previous one (Fig. 2) due to inactivation ofpolymerase activity during incubation with CIP or BSA (datanot shown). The higher activity of the CIP-treated RNApolymerase 110 was due to dephosphorylation of the largestsubunit of the enzyme because (i) the complexes formed withthe CIP-treated enzyme comigrated on a gel mobility shiftassay with complexes formed with the HA enzyme (Fig. 3B,compare lanes 7 and 8 with lane 3), and (ii) the CIP-treatedenzyme comigrated on an SDS/PAGE gel with the largestsubunit ofRNA polymerase 11A as detected by silver staining(Fig. 3C). Thus, these results (Figs. 2 and 3) demonstrate thatthe nonphosphorylated form of RNA polymerase II prefer-entially associates with the preinitiation complex.

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FIG. 4. Conversion of RNA polymerase IIA to the 110 form bya cellular protein kinase. (A) DNA binding reactions were performedas described in Fig. 2. Lanes 1 and 2 represent the DA and DABcomplexes, respectively. Lane 3 represents products of reactionsobserved in the presence of TFIIA, -1ID, -hIB, and -IIF and RNApolymerase 110 (140 ng). Lanes 4 and 5 are the same as in lane 3, butthe RNA polymerase II was the IIA (40 ng) and reactions wereperformed in the absence or presence of Hk1. (B) DNA proteincomplexes were formed as in A but ATP was omitted or substitutedby other ribonucleoside triphosphates (1 mM), as indicated. (C)Proteins present in the DNA-protein complexes formed with eitherRNA polymerase IIA (lane 1), RNA polymerase 110 (lane 2), nopolymerase but in the presence of a protein kinase (lane 3), or RNApolymerase IIA and protein kinase (lane 4) were extracted from thecomplex and separated by SDS/PAGE. The form of the enzyme inthe complex was analyzed by Western blotting using the enhancedchemiluminescence technique and antibodies directed against exon5 of the largest subunit ofRNA polymerase II as described in Fig. 2.

DAB- axM_ 2_m --3 c

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present in the carboxyl terminus of the largest subunit of theenzyme (5, 6, 14). Our studies resulted in the isolation of eachform ofRNA polymerase to apparent homogeneity. Using thepurified forms of RNA polymerase II, we found that the IIAform associates four times more efficiently than the 11O formwith a preinitiation complex formed at the Ad-MLP. Using gelmobility shift assays, we observed that the IIA form associatedfaster than the ITO form with the preinitiation complex. Ourstudies demonstrated that complexes formed with the IIAform contained the nonphosphorylated form in the complex.Conversion of the enzyme to the 11O form required a proteinkinase. The kinase activity required an RNA polymerase II inassociation with the preinitiation complex. Together all theseresults allowed us to conclude that the nonphosphorylatedform ofRNA polymerase II preferentially associates with thepreinitiation complex. Our findings could not be explained bypitfalls in the method used to isolate the 11O form, as we foundthat conversion of RNA polymerase 11O to the IIA form byphosphatase treatment in vitro resulted in the recovery ofRNA polymerase II that associated more efficiently with thepreinitiation complex. This was correlated with conversion ofthe enzyme to the dephosphorylated form. It is our belief thatIIO-mediated association with the preinitiation complex ispossible only in vitro conditions where excess, nonphysiolog-ical RNA polymerase levels are attainable.Our findings demonstrating that the nonphosphorylated

form of RNA polymerase II preferentially recognizes thepreinitiation complex are in perfect agreement with thestudies of Laybourn and Dahmus (17, 19) suggesting thatphosphorylation of the CTD regulates the transition frominitiation to elongation. This model is further supported byfindings demonstrating that the phosphorylated form ofRNApolymerase II preferentially associates with elongation com-plexes in isolated nuclei and in vitro (17, 19) and by ourobservation that a protein kinase can phosphorylate RNApolymerase II in association with the preinitiation complex.Similar results were obtained by Laybourn and Dahmus (19);however, their studies suggested that phosphorylation of theCTD was mediated by one of the general transcriptionfactors. Our studies indicate that phosphorylation ofthe CTDis catalyzed by a protein kinase distinct from the basaltranscription factors. Indeed our studies resulted in theisolation of two different protein kinases from HeLa cellsphosphorylating the CTD (H.L. and D.R., unpublished re-sults). In agreement with the studies ofothers (28, 33, 34), oneof these kinases copuriflies with cdc2 and cyclin B (H.L. andD.R., unpublished results).A great deal of controversy exists regarding the role of the

CTD of the largest subunit of RNA polymerase II (6). Ourstudies strongly suggest that phosphorylation of this domaincould at least in part regulate the transition from initiation toelongation. Our studies do not rule out a possible role for thismotif in mediating response to activators as suggested byAllison and Ingles (21). Work using highly purified compo-nents of the transcription machinery may provide some oftheanswers to the mystery behind the CTD of the largest subunitof RNA polymerase II.

This work was supported by grants to D.R. from the NationalInstitutes of Health, the National Science Foundation, and theAmerican Cancer Society. O.F. was supported by a fellowship fromSchering Corp. D.R. was a recipient of an American Cancer SocietyFaculty Research Award.

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