Regulation of the human general transcription initiation factor TFIIF by ...

THE JOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 47, Issue of November 25, pp. 29970-29977, 1994 Printed in U.S.A.

Regulation of the Human General Transcription Initiation Factor TFIIF by Phosphorylation* (Received for publication, April 11, 1994, and in revised form, August 12, 1994)

Shigetaka KitajimaS, Taku Chibazakura, Masatomo Yonaha, and Yukio Yasukochi From the DeDartment of Molecular Genetics, Medical Research Institute, lbkyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan

The transcription initiation factor,TFIIF, is essential quent incorporation of other factors, including TFIIE, TFIIH, not only for the initiation of transcription but also for and TFIIJ, forms the complete preinitiation complex (15). Of efficient elongationof mRNA synthesis by mammalian these reactions, the first step which includes TFIIA, TFIID, in and TFIIBhas been regarded as a major controltarget of action RNApolymerase I1 and is extensively phosphorylated vivo. The possible regulation TFIIF of activity by protein by several gene specific transcription factors. The molecular phosphorylation was investigated by comparingthe bio- mechanism of the action is proposed to be via protein-protein chemicalproperties of alkalinephosphatase-treated interactions between some of the general factors and gene HeLa TFIIF withthoseofnative or bacterially ex- specific factors (20-24). pressedfactor.AlkalinephosphatasetreatmentdeGene expression can also be regulated by modification of the creased thesize of the large subunit (RAP741 of TFIIFto specific factors. Some putative specific factors have been well that of the recombinant protein but did not change the studied in terms of regulation by protein phosphorylation of size of the small subunit (RAPSO). Both the transcription their nuclear translocation, DNA binding, or transactivation initiation and elongation stimulating activities of the properties (for review, see Ref. 25). Such control mechanisms alkaline phosphatase-treated TFIIF decreased to15-20% could exist for the general transcription machinery of RNA of the nativeform underconditions in which amount the ofTFIIFwas rate-limiting for transcription. Further- polymerase 11. However, the modification of the generalfactors more, phosphatase-treated TFIIF assembled the DBPolF in vivo and its significance remain unresolved. TFIIF (26,27), also termed FC(281, RAP30/74 (291, in rat complex and bound RNA to polymerase I1 less efficiently than the native protein. When hybrid TFIIFs were re- (30), or factor 5 in Drosophila (31), canbind RNApolymerase I1 constituted using native or recombinant subunits,a na- directly (26, 28, 29) and recruits the enzyme to a preformed tive form of RAP74 stimulated both transcription and DAB complex. Furthermore, TFIIF also stimulates theelongaDBPolF complex formation activity regardless of tion of nascent mRNA by RNA polymerase I1 (26,31,32). Thus, whether native orrecombinant RAP30 was used. We TFIIF is not only an initiation factor but also an elongation propose that TFIIF activity is regulated by protein phos- factor and can be a target of regulation at both of these stepsof RAP74 subunit. The mRNA synthesis. phorylation,particularlyofthe functional roleof RAP74 in assembling the preinitiation TFIIF isa heterotetramer composed of a small (RAP30) and complex and modulating TFIIFactivity is discussed. a large (RAP74) subunit (27, 28, 29), both of which are phosphorylated in vivo (26, 29, 33). Native TFIIF subunits have a lower mobilitythan the bacterially expressed recombinant proThe synthesis of mRNA in eukaryotes is carried outby the teins on SDS-PAGE (341, indicating that TFIIF is posttranslaRNA polymerase I1 transcription machinery. RNA polymerase tionally modified in vivo. In this article, we investigate the I1 is a large multisubunitenzyme that nonspecifically catalyzes significance of the modification of TFIIF by protein phosphorylation by assaying thebiochemical properties of HeLa native, the synthesis of mRNA from a DNA template, but requires additional proteinfactors, commonly referred toas the general dephosphorylated, and bacterially expressed TFIIF. We report or basal factors, to initiate transcription accurately at promot- that modification of the RAP74 subunit by protein phosphorylof TFIIFbinding to the RNA ers (1-6). Thegeneralfactors include transcription factor ationregulatestheafinity polymerase 11, thereby modulating the activity of TFIIF inboth TFIIA,' TFIIB, TFIID, TFIIE,TFIIF, TFIIH, and TFIIJ(7-141, but additionalfactors might exist.Among these factors, TFIIA, the initiation andelongation reactions of transcription. TFIID, and TFIIB first recognize the promoter sequence of the MATERIALS AND METHODS RNA polymgene (15-17) and, subsequently TFIIF along with Zhnscription Factors-HeLacell cultures and the preparation of erase I1 enters the preformed DAB complex (18, 19). Subsenuclear extracts were performed as described by Dignam et al. (35),and (11). the fractionation procedure was carried out as described previously * This work was supported by a grant-in-aid for Scientific Research The activities of FD and FA could be replaced by bacterially expressed of Japan and by a TBP and TFIIB, respectively, indicating from the Ministry of Education, Science, and Culture that FD and FA correspond to grant from the International Human Frontier Science Program Orga- TFIID and TFIIB, respectively. The FE fraction was shown to contain nization. The costsof publication of this article were defrayed inpart by two factors that were separated on a phosphocellulose (P11)column. the payment of page charges. This article must therefore be hereby The FE fraction seer DEAE-Toyo and S-300HR column chromatogramarked"advertisement"inaccordancewith 18 U.S.C.Section 1734 phies was applied onto a P11 columnequilibrated with bufferB (20 mM solely to indicate this fact. Tris-HC1 (pH7.9), 0.1 mM EDTA, 20% glycerol, and 5 mM P-mercapto$ To whom correspondence should be addressed: Dept. of Molecular ethanol) containing0.1 M NaCI. It was then eluted by a linear gradient Genetics,MedicalResearch Institute, TokyoMedical andDental University, 1-5-45Yushima, Bunkyo-ku, Tokyo113, Japan. Tel.: 3-3813- of NaCl from 0.1 to 0.7 M. Two overlapping peaks of activity complementing FE were observed at 0.3 and 0.36 M NaCI, respectively. The 6111 (ext.) 6141; Fax: 3-3813-8684. The abbreviations used are: TF, transcription factor; PAGE, polyac- latter peak of activity contained the 33-kJ3a protein as reported by us (36) and was further purifiedon a QAE-Toyocolumn.The purified rylamidegelelectrophoresis;BSA,bovine serum albumin; bp,base fraction that was eluted from the column at 0.36 M NaCl in buffer B pairs; A P , alkaline phosphatase.

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Phosphorylation of HeLa TFIIF contained the 55-and 33-kDa proteins as major constituents, and peptide analysis of the 33-kDa protein yielded the same partialsequence of amino acids as thatof the small subunit of TFIIE (37, 38), indicating that the factor was TFIIE. The first peak of FE activity on the PI1 coluan was further purified on Q-Sepharose and S-Sepharose columns and found to be essential in the in vitro transcription assay reconstituted with the TFIIE purified as above. The PAGE pattern of the factor resembled that of BTF2 (TFIIH) reported by others (13, 14), although further purification must be done in order to make a final identification. FF, apparently equivalent to TFIIA in chromatographic and kinetic characteristics (12,39), was no longer essential in the complementation assay as our purification proceeded. FF, however, can still stimulatethe in vitro transcription activity by 2-3-fold in our assay. TFIIF, our original FC, was purified to homogeneity as before (28) with minor modifications. Briefly,TFIIF was eluted from a P11 column by a linear NaCl gradient from 0.3 to 0.6 M in buffer B with a peak of elution a t 0.42 M. After Sephacryl S-3OOHR column chromatography of the P11 fraction as before (281, the active TFIIF fraction was applied to a Q-Sepharosecolumn equilibrated with buffer B containing 0.2 M NaCl and eluted by a linear gradient from 0.2 to 0.6 M. The TFIIF activity eluted at 0.32 M NaCl and appeared homogeneous. This modified procedure yielded 34-fold higher recovery of protein in pure form than when using the DEAE high performance liquid chromatography column as in (28). Our FB is still difficult to purify and cannot be compared with any factor reported by others. FB was shown to be essential in our reconstitution assay of transcription (11) and probably contains TFIIJ, TFII-I, and other unidentified factors. Recombinant TFIIF was obtained by expressing pETRAP30 or pETRAP74 in BL21(DE3) cells and purified as before (34,40). TBP was expressed in BL21(DE3) cellsharboring pETHIID (41) and purified by successive chromatography on P11 and DEAE-Toyo columns. The final TBP was at least 90% pure on a protein gel and active in an FD complementationassay. TFIIB was also obtained by expression of phIIB in BL21(DE3) cellsas described (42) and purified on P11, S-Sepharose, and single-stranded DNA columns. The recombinant TFIIB protein was over95% pure and catalytically active in our FA complementation assay. RNA polymerase I1 was purified from calf thymus or HeLa cell nuclear pellets by the procedures of Hod0 and Blatti (43) or Reinberg and Roeder (7), respectively. Beatment ofHeLa TFIIF by Alkaline Phosphatase-HeLa TFIIF (0.4 pg) was incubated in a total of 10 pl of 50 mM Tris-HC1 (pH 7.9) containing 1 mM MgCl,, 5 mM DTT, 1 pg of BSA, and in the presence or absence of0.25 unit of calf intestine alkaline phosphatase (Takara, Kyoto, Japan) at 30 "C for various time periods. Each aliquot was assayed for TFIIF activity after the addition of 20 mM NaF or analyzed on SDS-PAGE. To study the binding of alkaline phosphatase-treated TFIIF with calf thymus RNA polymerase 11, HeLa TFIIF (5 pg determined by Western blot, S-3OOHR step) was treated with 2.5 units of alkaline phosphatase at 30 "C for 30 min. After adding 5 of 1M NaF to a final concentration of 20 mM and 50 pg of calf thymus RNA polymerase 11, the mixture was dialyzed against buffer B containing 0.1 M NaCl and 20 mM NaF at 4 "C for 8 h. The mixture was applied onto a Sephacryl S-300HR column(1.4 x 46 cm) equilibrated with the dialysis buffer. Protein elution was monitored by W absorbency and 0.32 mUfractions were collected. Successive fractions beyond the void volume were combined and vacuum-dried to 100 p1. Reconstitutionof Hybrid TFIIF-Native HeLa TFIIF (S-300HRstep, 50 pg determined by Western blot analysis) was separated on a 10% SDS-PAGE and stained with 4 M sodium acetate. Both subunits of TFIIF, RAP30 and RAF'74, were excised and eluted from the gel into 0.5 ml of 20 m~ Tris-HC1(pH 7.91,O.l mM EDTA, 5 m~ DTT, 0.1 mg/mlBSA and 0.15 M NaCl by rotary shaking overnight. The eluted subunit was precipitated with acetone, rinsed twice with 80%acetone, and dissolved in 30 pl of 8 M urea. After 0.5p1 of each subunit was separated by a 10% SDS-PAGE along with HeLa TFIIF and recombinant TFIIF subunits and transferred onto a nitrocellulose membrane, the amount of each protein was determined by Western blot.Apparently, equimolar amounts (equivalent to 2 pg of ILAP30) of each TFIIF subunit,native or recombinant, were mixedin 10 111 of 8 M urea. After dilution to 50 pl by buffer B containing 0.1 M NaCl and 10 pg ofBSA, the mixture was dialyzed against buffer B containing 0.1 M NaCl using a microdialyzer (Pierce Chemical Co.) at 4 "C overnight. In Vitro Banscription Assay-The in vitro transcription activity of TFIIF was assayed as described in Ref. 11, and RNA transcripts were analyzed on 6% polyacrylamide, 7 M urea gels. The reaction was performed in a total of 30 pl containing 10 ng of TBP, 20 ng of TFIIB, 0.8

pg of FB, 0.4 pg of FE (a mixture of TFIIE and TFIIH as described above), 20 ng of TFIIF, and 0.5 pg of HeLa RNA polymerase I1 using 2 pg of plasmid containing the adenovirus major late promoter gene.The template DNAs used werepMLC2AT (44) or pAd2 (11)for the freeround or Sarkosyl-blockedsingle-round assay, respectively. Forthe freeround transcription assay, the above mixture was incubated for the assembly of the preinitiation complex at 29 "C for 30 min, and nucleoside triphosphates were added to a final concentration of 600PM for ATP and UTP and 25 VM for [n-32PlCTP(5 pCi). After 60 min, the reaction was stopped and radioactive RNA was isolated and analyzed. For the single-round assay, the above mixture was first incubated at 29 "C for 40 min. One min after initiating transcription by adding a mixture of 600 p ATP, UTP, and 25 p~ [CC-~~PICTP (5 pCi), Sarkosyl was added t o a final concentration of 0.25%. Onemin later, 10 mM GTP was added to a final concentration of 600 p,and elongation was allowed to proceed for another 40 min. Transcription activity was quantitated by measuring the densities of the transcripts by densitometry and expressed as arbitrary units. Gel Mobility Shift Assay-DBPolF complex formation was measured in a volume of 30 pl containing 10 mM Tris-HC1 (pH 8.0), 50 mM NaC1, 10% glycerol, 5 m~ MgCl,, 1m~ DTT, 0.05 mM EDTA, 0.2 mg/ml BSA, and 200 ng of poly(dG.dC).poly(dGdC).40 ng of TBP was incubated with a radiolabeled promoter fragment at 30 "C for 30 min, followed by the addition of a mixture of 30 ng of TFIIB, 50 ng of TFIIF, and 500 ng of HeLa RNA polymerase 11. After incubation for another 30 min, the reaction mixture was loaded ontoa 4% polyacrylamide gel (acrylamide: bis = 40:l) containing 2.5% glycerol in a buffer of 40 mM Tris borate, 1 mM EDTA. After electrophoresis at room temperature, thegel was fixed in methanoyacetate, vacuum-dried, and autoradiographed. The promoter fragment for the assay was purified by digestion with BamHI and Hind111 of the plasmid pAD2TATA containing the promoter sequenceof adenovirus type 2 major late promotor gene from -55 to +33. The fragment was labeled with [ C Y - ~ ~ P I and ~AT Klenow P enzyme, and 2 ng of the labeled fragment (10,000 cpm) wasused in each assay. Assay of Elongation of mRNA Synthesis by RNA Polymerase II-The elongation activity of mRNA synthesis by RNA polymerase 11 was assayed using purified HeLa RNA polymerase I1 and a poly(dC)-tailed DNA template as described (45). To prepare the poly(dC)-tailedtemplate, the plasmid pBR322 was digested with PstI and poly(dC)tails of 3 5 4 0 nucleotides were added to the3' ends using dCTP and terminal deoxynucleotide transferase. After digestion of the DNA with Ssp1 to generate two fragments (0.56 and 3.8 kilobase pairs), the 3.8-kilobase pairs fragment (termed dC3.8) was purified on an agarose gel and used as a template. The elongation assay was performed in a total of 20 pl containing 16 m~ Tris-HC1 (pH 7.9), 80 m~ NaC1,16% glycerol,0.08 mM EDTA, 5 m~ MgCl,, 1 mM DTT,and 0.1 unit of RNase inhibitor. HeLa RNA polymerase I1 (0.05 pmol) and dC3.8 template DNA (0.1 pmol) were first incubated in the presence or absence of TFIIF at 25 "C for 5 min. Transcription from dC3.8 was initiated by the addition of 0.5 m~ each ATP, GTP, CTP, and 0.25 p [CC-~~PIUTP (10 pCi). Afterincubation for 10 min, the initiated RNA transcripts were elongated by adding 0.5 mM unlabeled UTP for various time periods, and the reaction was stopped with 200 pl of 0.5% SDS and 10 mM EDTA. The labeled RNA transcripts were recovered and resolved on a 6% polyacrylamide gel containing 8 M urea. Other Procedures-SDS-PAGE was performed as in Laemmli (461, and the gels were stained by two-dimensional-SILVERSTAIN I1 from Daiichi. Immunoblotting was performedwith Western Light from Boehringer Mannheim. The first antibody was rabbit anti-RAP30 or antiRAP74 (34) and the second antibody was alkaline phosphatase-conjugated goat anti-rabbit IgG. Quantitative measurements of the x-ray bands were performedby using a densitometer (MolecularDynamics)or an image analyzer (Fujix BAS2000). Calf intestine alkaline phosphatase, terminal deoxynucleotidetransferase, Klenow enzyme, and RNase inhibitor, were purchased from Takara (Kyoto, Japan). All other materials were reagent grade. RESULTS

Effects of Alkaline Phosphatase Deatment-We have reported previously that both bacterially expressed RAP30 and RAP74 have smaller molecular sizes on SDS-PAGE than do the subunits of native TFIIF, suggesting that TFIIF is modified in vivo (34).To study the effect of possible modificationby protein phosphorylation, TFIIF purified from HeLa cells was treated with calf intestine alkaline phosphatase in vitro. As shown in Fig. 1, the treatment resulted in a decrease of the size of the

Phosphorylation of HeLa TFIIF

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1 2 3 4 5 6 7 8 9 1 0 FIG.1. SDS-PAGE analysis of TFIIF dephosphorylated in vitro by calf intestine alkaline phosphatase. Purified or S-300HR fraction of HcLa TFIIF was treated with the indicated amount of calf intestine alkaline phosphatase ( A P ) for 30 min as described under “Materials and Methods.” Each aliquot (1 1.11, 40 ng of TFIIF) of the reaction was resolved by 10% SDS-PAGE, and the proteins were visualized by silver staining.Lunes 1 and 10,purified TFIIF; lune 2, TFIIF treated with 0.25 unit of alkaline phosphatase; lune 3,same as inlune 2 but in the presence of 20 my NaF; lunes 4-8, S-300HR fraction of TFIIF treated with 0, 0.05, 0.1, 0.25, and 0.5 unit of AF’, respectively. Lune 9, a mixture of bacterially expressed RAP30 and RAP74. The upper t w o urrows indicate the positionsof the native and recombinant RAP74, respectively, and the lower urrows indicate those of the native and recombinant RAp30, respectively.

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RAP74 subunit of IIF, but not of the RAP30 subunit (lanes 1 and 2 ) . The mobility of thealkalinephosphatase-treated RAP74 was indistinguishable from that of the recombinant protein (compare lanes 2 and 8 with 9 ) , indicating that the difference in the size of RAP74 is mainly due to phosphorylation. That the observed effect was due to the phosphatase activity but not due to some proteolytic activity contaminating the enzyme preparation was supportedby the fact that20 mM NaF, a phosphatase inhibitor, could totally inhibit the effect ( l a n e s 2 and 31, whereas the addition of protease inhibitors, including phenylmethylsulfonyl fluoride,leupeptin, andbenzamidine, had no effect (data not shown). When a crude preparation of TFIIF (S-3OOHR step) wasdigested with the enzyme, some, but not all, other proteins also changed their mobilities ( l a n e s 4-8), suggesting that only the phosphorylated proteins were substrates of the reaction. Furthermore,the reaction using recombinant RAP74 protein radiolabeled in vitro with [y-:lzPIATPand cyclic AMP-dependent protein kinase resulted in the releaseof radioactivity (data not shown). We next assayed the in vitro transcription activity of the alkaline phosphatase-treated factor using the adenovirus major late promoter gene in both the free-round (Fig. 2 A ) and Sarkosyl blocked single-round (Fig. 2B ) assays. Thefree-round assay produced a 390-nucleotide transcript from the fusion gene of the adenovirusmajor late promoter and G-less cassette (pMLC2AT). The transcript in theSarkosyl-blocked assay was 186 nucleotides in lengthfrom the native adenovirus major late promoter gene (pAd2)as reported previously (11). Fig. 2, A and B, clearly show that phosphatase treatment for 60 min diminished the activity in both assays approximately 80% compared with the minimal reduction in the control (20%) that was incubated without alkaline phosphatase. Another control reaction in which TFIIF was incubatedwith alkaline phosphatase

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FIG.2. In vitro transcription activity of dephosphorylated TFIIF. Aliquots (1 pl, 40 ng) of the purified TFIIF treated with 0.25 unit alkaline phosphatase ( A P )for various time periods were assayed for in vitro transcription activity aRer the addition of 20 mM NaF as described under “Materials and Methods.” A, an autoradiogram of the gel for the free-round transcription products. Lanes 2 3 show control TFIIF treated in the absence ofAF’ for0 (lune 2 ) , 15 (lune 3). 30 (lune 4).and 60 min (lune 51, respectively; lunes 6 9 were treated with AP for 0 (lune 6 ) ,15 (lune 7), 30 (lune8 1, and 60 min (lune9), respectively. For 0-min treatment (lune 6 ) , AF’ and 20 m~ NaF were added simultaneously. Lane 1 shows the activity without the addition of TFIIF. B, results of the Sarkosyl-blocked singleround assay of TFIIF dephosphorylated as inA. Lanes 1 4 were those with control TFIIF treatedwithout alkaline phosphatasefor 0 (lune 1 ), 15 (lane 2), 30 (lune 3 ) , and 60 min (lune 41, respectively, and lunes 6 9 were those with TFIIF treatedwith alkaline phosphatasefor 0 (lune 6), 15 (lune 7), 30 (lune8),and 60 min (lune9), respectively.Lanes 5 and 10 show the activity without TFIIF. C, the transcripts of the control and the alkalinephosphatase-treated TFIIF in A and B were quantitated by densitometer and plotted as a percentage of the value at 0 min for the free-round (0,O) and Sarkosyl-blocked single-round (El,W)assay. Open and closed symbols represent activities of the control and thealkaline phosphatase-treatedTFIIF, respectively. D,five times more of the control and alkaline phosphatasetreated TFIIF (200 ng) treated as in A were assayed for the free-round transcription and theautoradiogram of the gel is shown. Lane 1, no addition of TFIIF; lune 2, control (0rnin); lune 3, control (60 min); lune 4, alkaline phosphatase-treated(0min);lune 5 , alkaline phosphatase-treated (60 min).

and 20 mM NaF as in Fig. 1, lane 2, also retained full activity, providing further evidence that theeffect is due to the removal of phosphate from TFIIF (data not shown). Comparison of the activities in both assays, free-round and single-round, showed


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FIG.3. DBPolF complex formationby dephosphorylated DBPolF complex was detectedon a 4 0 native acrylamidegel a s described under “Materials and Methods.”A, the binding reaction included the following components: lane 1, no addition; lane 2, 40 ng of TBP alone;lane 3,40ng of TBP and 30 ng of TFIIB; lane 4 , 40 ng of TBP, 30 ng of TFIIB, and 500 ng of RNA polymerase 11; lanes 5-7, same as in lane 4 but containing 50 ng(lane 5 ) , 15 ng (lune 6 ) ,and 5 ng of TFIIF (lane 7), respectively. The upper arrow indicates thespecific DBPolF complex, and the lower arrowshows a nonspecific complex between DNAprobe and RNApolymerase 11. In B , 0.4 pl(16 ng)of the TFIIF alkaline phosphatase-treated as in Fig. 2 was assayed for the DBPolF complex formation activity. Lanes 1 and 2, treated without alkaline phosphatase for 0 and 60 min, respectively; lanes 3-6, treated with alkaline phosphatasefor 0 (lane 3),15 (lane 4 ) . 30 (lane 5 ) , and 60 min(lane 6 ) ,respectively. In C , DBPolF 3 pl(120 ng) of TFIIF treated without alkaline phosphatase for 0 (lane 1 ) and 60 min(lane 2 ) or treated with complex formation was assayed using alkaline phosphatase for 0 (lane 3)and 60 min (lane 4 ) . Treatment with AP for 0 min was an incubation in the presenceof 20 mM NaF.

that our reconstitutedfree-round transcription assay could not specific and represented theDBPolF complex, since it could be reinitiate (data not shown), indicating that the reinitiation observed after TBP, TFIIB, RNApolymerase 11, and TFIIFwere transcription factor described by Szentirmay and Sawadogo sequentially added to the binding reaction (Fig.3A, lanes 2 5 ) . (47) is present at very low levels in our assay mixture. Thus, It was notdetected when either TBP, TFIIB, RNA polymerase not only the Sarkosyl-blocked assay but the free-round assay of 11, or TFIIFalone was removed from the complete reaction (data our reconstituted transcription represents the amount of active not shown). In our assay, the intensity of the DBPolF complex preinitiation complex. This is consistent with our observation was significantly enhanced compared with that of the DB that the alkaline phosphatase-treated TFIIFshowed a compa- complex which is unstable in the binding buffer containing rable decrease of the activities in both assays (Fig. 2C). Fig. 20 NaClinstead of the KC1 employed by others (15, 18, 191, indishows the activitieswhen assayed usingfive times more of the cating that the recruitment of RNA polymerase I1 by TFIIF is factor. Under this condition, the alkaline phosphatase-treated synergistic and greatly stabilizes the resulting DBPolF complex. TFIIF exhibited comparable activity with that of the control. Fig. 3B shows that the treatment of native TFIIF with phosAn immunoblot analysis of the alkaline phosphatase-treated phatase decreased the DBPolF complex formation to 20-30% of TFIIFseparated by SDS-PAGE showed that over 95% of the control, comparable with the extent of reduction of the in RAP74 shifted itsgel mobility to thatof the recombinantfactor vitro transcription activity (Fig.2, A-C). The time course profile after a 60-min incubation (data not shown). Therefore, the al- of the decrease in thecomplex forming activityof TFIIF by the kalinephosphatase-treatedTFIIFhas reduced activitybut phosphatase treatmentalso resembledthat of the invitro tranlarger quantities of the factor can rescue the activity. The scription activity (data not shown). However, when 7.5 times residualamount of phosphorylated TFIIF was too small a more of the factor was assayed, it produced as much of the fraction of thetotalmaterial to entirely account for the DBPolF complex as that of the control (Fig. 3C). These data observed activity. strongly indicate that the decreased transcription activity of the Zn Vitro Assembly Analysis-The assembly of the transcrip- alkaline phosphatase-treated TFIIF is due a t least partly to the tion initiation complex formed by the promoter sequence of the reduced ability to assemble the active DBPolF complex. gene and transcription factors has been analyzedby a native gel Activity of Hybrid TFZZF-TFIIF activity requires both submobility shift assay(15,18, 19). TFIIF hasbeen reported to be units, RAP30 and RAP74, and has been reconstituted by each essential in recruiting RNApolymerase I1 to thepreformed com- subunit separated from HeLa native TFIIF (27-29) or bacteriplex comprised of the promoter DNA,TBP, and TFIIB (DB ally expressed recombinant protein (40,48).In order toclarify complex) (18, 19). We employed this method to assess theeffect the role of the modification of each subunit in regulating the of phosphatase treatment on the function of the TFIIF in the TFIIF activity, hybrid molecules comprised of each subunit assembly of the preinitiationcomplex using thepromoter DNA were reconstituted as described under “Materials and Methsequence of the adenovirusmajor late gene. As shown in Fig. 3A, ods,” and testedfor their in vitro transcription activity. Fig. 44 the additionof RNA polymerase I1 along with TFIIF to the DB shows the preparationof each subunit of HeLa or recombinant complex produced a high molecular weightband. The band was TFIIF. Fig. 4, B and C, show the results of the free-round in


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hybrid IIF ng

FIG.4. In vitro transcription activity of hybridmolecules of TFIIF reconstituted from native or recombinant TFIIF subunits. The hybrid TFIIFs were reconstituted using gel-purified subunits of native HeLa TFIIF and recombinant subunits described as under "Materials and Methods." SDS-PAGE of each subunitfor the reconstitution isshown in A. Lane 1 , 4 0 ng of native TFIIF;lane 2,30 ngof gel-purified nativeRAP74; lane 3,20ng of bacterially expressedRAP74; lane 4,20ng of gel-purified nativeRAP30; lane 5,20ng of bacterially expressedRAP30. B , the hybrid TFIIFs were assayedfor the free-round in vitro transcription activity using increasing amounts of the factors. Lane 1, no addition of TFIIF; lane 2,150 ng recombinantRAP30 (r30);lane 3,150ng of recombinant RAP74 (r74); lanes 4-7,15 ng (lane 4).40 ng (lane 5),100 ng(lane 6 ) ,and 150 ng (lane 7 )of the hybrid of native RAP30 (n30)and native RAP74 (n74); lanes 8-11,15 ng (lane 8),40 ng (lane 9 ) , 100 ng (lane IO),and 150 ng (lane 11)of the hybrid of n30 and r74; lanes 12-15, 15 ng (lane 12),40 ng (lane 13),100 ng (lane 14),and 150 ng (lane 15)of the hybrid of r30 and n74; lanes 16-19,15 ng(lane 16),40 ng (lane 17),100 ng (lane 18),and 150 ng (lane 19) of the hybrid of 1-30 and r74. In C , the transcripts of the hybrid TFIIFs comprised of n30 and n74 (O),n30 and 1-74 (O),r30 and n74 (A), and r30 and r74 (A),respectively, were quantitated by densitometer and plotted against each amountof the hybrid in arbitrary units.

vitro transcription assay using increasing amounts of these hybrid TFIIFs. It is evident that any combination of both subunits, whether nativeor recombinant, reconstituted theactivity. However, the efficiency with which TFIIF activity was reconstituted differed markedly among the hybrids. When assayed using a small amount of the hybrid factor, the TFIIF comprised of nativesubunits yielded thehighest activity, whereas the mixture of both recombinants showed only 1520% of the activity of the native reconstituent(compare lanes 4 and 5 with 16 and 17).The hybrid TFIIF comprised of the native RAP30 and recombinant RAP74 was no more active than the recombinant TFIIF. By contrast, thecombination of the recombinant RAP30 and native RAP74 was four to five times more active than that of the recombinant subunits and showed almost the same activity as the native TFIIF(lanes 12 and 13). However, the assays using larger amounts of the hybrid TFIIFs all reached a comparable level of transcription activity. We next investigated the DBPolF formation activity of the hybrid TFIIF. Fig. 5 shows that the TFIIFof both native subunits yielded the highestactivity (lane 21, whereas the TFIIFs of other combinations, the native RAP30 and recombinant RAP74, or the recombinant TFIIF showed minimal complex formations (lanes 3 or 5).By contrast, the TFIIFcomprised of the recombinant RAP30 and nativeRAP74 showed the activity comparable withthat exerted by the native reconstituent(lane 4 ) . However, the difference in the activity of the DBPolF formation was evident only when small amounts of TFIIF (15 ng) were used. As shown in lanes 7-10 of Fig. 5, there was no apparent difference in the activity of the complex formation when larger amountsof the hybrid TFIIFs (100 ng) were used. This is consistent with the results of the in vitro transcription assay (Fig. 4, B and C).The combined results clearly indicate that theRAP74 subunit hasa stimulatory function that is most evident at suboptimal assay conditions for both transcription and DBPolF complex formation activities. Furthermore, the activity of RAP74 is affected by posttranslational modification in vivo.

-

n30n30 r30 r30 n74 r74 n74 r74

-

n30n30 r30 r30 n74 r74 n74 r74

f-

1

2

3

4

5

6

7

8

9

1

0

FIG.5. DBPolF complexformation activity of the hybrid TFIIFs. Assay for the DBPolF complex formation activityof the hybrid TFIIFs was performed as described under "Materials and Methods" using 15 ng (lanes 2-51 or 100 ng (lanes 7-10)of the factors. Lanes 1 and 6, no addition; lanes 2 and 7, the n30 and n74 hybrid; lanes 3 and 8,the n30 and 1-74hybrid; lanes 4 and 9, the r30 and n74hybrid; lanes 5 and 10,the r30 and r74 hybrid. The arrow indicates the specific DBPolF complex.

Elongation Stimulation Activity andPhysical Interaction of the Alkaline Phosphatase-treated TFIIF with RNA Polymerase 11-TFIIF also stimulates the elongation reaction ofmRNA


n

(nt)

720

control 0‘ 60‘

0‘

AP 15’ 30‘

A

29975

D

60‘

31

1. “2. .3

~!zLn“!z-I I I I

-

535 420 311 -

“ W

249 200

-

fraction

B

151 140

118 -

100 -

1 2 3 4 5 6 7 8 9 1011 121314

t-

82 -

---

66 ”

321 4 5 6 7 8 9 1 0 1 1 1 21 3 1 4 FIG.6. Stimulation of elongation of mRNA synthesis by dephosphorylated TFIIF. 1.5 pl(60 ng) eachof TFIIF dephosphorylated as in Fig. 2 was assayedfor its ability to stimulate elongation of mRNA synthesis by RNA polymerase I1 according to the procedure described under “Materials and Methods.” Lanes I and 2, no addition of TFIIF; lanes 3-6, TFIIF treated without alkaline phosphatase for 0 min (lanes 3 and 4 )and 60 min (lanes 5 and 6); lanes 7-14,treated with alkaline phosphatase in the presence of 20 m~ NaF (lanes 7 and 8)or without NaF for 15 min (lanes 9 and IO),30 min (lanes I1 and 12),and 60 min (lanes 13 and 14).Odd and even numbered lanes show the elongated transcripts after2- and 4-min reaction,respectively.

c

c

C

-

1 2 3 4 5 6 7 8 9 1011121314

c

synthesis by RNApolymerase I1 (26,31,32). To understand the effect of dephosphorylation of TFIIF on its elongation stimulation activity, we assayed mRNA synthesis initiated from the c artificial de-tailed DNA promoter by RNA polymerase I1 using both the control and the alkaline phosphatase-treated TFIIF as described under “Materials andMethods.” As shown in Fig. 6, the stimulatingactivity of the factor was also reduced by phosFIG.7. Binding affinityof alkaline phosphatase-treated TFIIF’ phatase treatment. Since the assay contained only TFIIF and for RNA polymerase 11. HeLa TFIIF (5 pg, S-300HR step) treated for its binding to 50 pg of RNA RNApolymerase I1 as theprotein factors,this suggests that thewith alkaline phosphatase was assayed polymerase I1 as described under “Materials and Methods.” Aliquots (10 interaction between TFIIF and RNA polymerase I1 could be pl) of the fractions eluted from the column were assayed for RNA poinhibited by phosphatase treatment, leading to a reduction of lymerase I1 (43) to detect the elution position of the enzyme (A) or the elongation stimulating activity. Therefore, we next investi- subjected to 10% SDS-PAGE, transferred onto a nitrocellulose memgated thebinding affinity of the alkaline phosphatase-treated brane, and immunoblotted with a mixture of antisera against RAP30 RAP74 to determine the elution position ofTFIIF for the control ( B ) TFIIF to RNA polymerase 11.We employed a gel filtration and and the alkaline phosphatase-treated factor(C). The Upper and lower method as described previously (281, since the glycerol gradient arrows indicate RAP74 and RAP30, respectively. In D,the two peaks of centrifugation did not yield good separation between the bound TFIIF in the experimentC were analyzed by 10% SDS-PAGE followed and free TFIIF. As shown in Fig. 7, the alkaline phosphatase- by immunoblotting with anti-RAP74antibody. Lane 1,bound fraction of control TFIIF; lanes 2 and 3, bound and unbound fractions of the treated TFIIF bound to RNA polymerase I1 with less affinity the alkaline phosphatase-treated TFIIF,respectively. The upper and lower than the control (Fig. 7, B and C). Approximately 80% of the arrows indicate the native and alkaline phosphatase-treated RAP74, alkaline phosphatase-treated TFIIFremained unbound under respectively. the condition where the control TFIIF was fully bound to the the factor dephosphorylated in vitro with alkaline phosphatase enzyme. The two peaks of the alkaline phosphatase-treated TFIIF, bound and free, respectively, were also analyzed on SDS- withthose of native or bacterially expressed recombinant TFIIF. 70). Theresult PAGEfollowedby Westernblotting(Fig. The apparentsize of the RAP74 subunit of TFIIF decreased showed that the RAP74 subunits in both peaks of TFIIF had became indistinguishable apparently the same molecular weight which is smaller than afterphosphatasetreatmentand that of the untreatedTFIIF, suggesting that both were dephos- from that of the recombinant protein, probably because of the phorylated t o the same extent.This indicates that the alkaline conformational changes induced by dephosphorylation of TFIIF phosphatase-treated TFIIF also binds to the enzyme with less (Fig. 1).The extent of dephosphorylation of TFIIF was not determined. The mobility change of RAP74 on SDS-PAGE was affinity than the native factor. observed with 0.1 unit of phosphatase defined by the supplier in DISCUSSION the standardassay, suggesting that thephosphorylated residue(s1 The significance of the phosphorylation of TFIIF, a general of the subunit iseasily accessible to the phosphatase treatment. human transcription initiation factor for RNA polymerase 11, In contrast, treatment with 1.0 unit of enzyme had no effect on the was investigated by comparing the biochemical properties of mobility of RAP30 (data not shown). RAP30 might have phospho-

29976


rylated residue($ which are highly resistant to digestion with (58)reported that the coactivator-like activity of serum rephosphatase in vitro or might contain modifications other than sponse factor o f the c-fos protooncogene is TFIIF and affects protein phosphorylation, since the native RAP30 subunit exhib- transcription by promoting initiation complex formation. They RAP74 subunit could bind DNA ited a larger size on SDS-PAGE than the recombinant RAP30 further demonstrated that the in conjunction with serum responsefactor and other DNA (Ref. 34, also see Fig. 1,lanes 9 and 10). Alkaline Phosphatase-treated TFIIF showed reduced activ- binding factors, including GAL4-vP16. Therefore, it is highly ity for i n vitro transcription, inboth the free-round and single- likely that RAP74 can also function in the assembly of the round assays (Fig. 2). The stimulation of elongation of mRNA preinitiation complex. We propose that RAP74 is also involved synthesis by RNA polymerase I1 was also reduced by the phos- inthe assembly step of thepreinitiation complex of the phatase treatment of TFIIF (Fig. 6). The kinetics of the tran- basal transcription, and its activity is regulated by protein scription activity of the dephosphorylated or reconstituted hy- phosphorylation. of We havereported previously the functionaldomains brid TFIIF showed that the reduced activity was evident only when a small amount of the factor was used. With a larger RAP74 which apparently correlate with itsproposed structure, amount of TFIIF, full activitywas seen, whether the factor was i.e. a globular N-terminal (1-179), a charged internal (180native, dephosphorylated, or bacterially expressed (Figs. 2and 3561, and a globular C-terminal(357-517) regions(34). Both N4). A piece of biochemical evidence that supports the kinetic and C-terminalregions werefound to be essential for transcription activity, the former of which was involved in associating differences was obtained by experiments that detected a dewith RAP30. TFIIF activity is probably regulated by this assocreased efficiency of the DBPolF complex formation (Fig. 5 ) or RAP74 the reduced affinity to bind to RNA polymerase I1 by the alka- ciation sincethe activity requires both subunits and the deletion that did not associate with RAP30 could not support line phosphatase-treated TFIIF (Fig. 7). The results were conthe transcription (34). Therefore, we speculate that RAP74, sistent with the idea that the protein phosphorylation of TFIIF particularly themodified form in vivo,increases theaffinity of up-regulates its activity by affecting boththe initiation and the RAP30 for RNA polymerase I1 or other component(s) of the elongation of transcription. Furthermore, the full recovery of DBPolF complex by modulating the heteromeric association, the transcription and DBPolF complex formation activities by even though each subunit functionsdifferently at in vitro tranthe hybrid TFIIF comprised of the recombinant RAP30 and scription initiation as proposed by Chang et al. (49). Another native RAP74 (Figs. 4 and 5 ) strongly indicates the regulatory possibility is that themodified RAP74 has anincreased activity function of the RAP74 in the process of active preinitiation of the C-terminal domain or interacts more efficiently with complex formation. It could not be determined whether the RNA polymerase I1 or TFIIB. different activities of the reconstituted TFIIFs resulted from Sopta et al. (33) originally described the phosphorylation of different levels of activity of the complexes that do form or the RAP30 and RAP74 in mouse erythroleukemia cells and obefficiency of active complex formation among the hybrids. In served that both the phosphorylated and dephosphorylated addition, there wasa n experimental limitation in the detection form of RAP30 can bind to RNA polymerase I1 using affinity of the physical binding of hybrid TFIIFs toRNA polymerase I1 columns containing immobilized enzyme. However, RAP74 was because the recombinant RAP30 aggregates as a large mole- still heavily phosphorylated in their experiment. cule, and RAP74 exists as tetramer in solution (34). Therefore, The RAP74 protein contains uniquesequences of amino acids we cannot rule out the possibility that the native form of at its internal domain of 180-356. This domain is proposed to RAP74 could interact more efficiently with RAP30 to form ac- provide the protein with theability to form a tetrameric intertive TFIIF.Recently, Chang et al. (49) reported that the RAP30 action as seen with native TFIIF (34). Multipleclusters of both subunit is sufficient to initiate i n vitro transcription from the positive and negative charges havebeen found along with sevadenovirus majorlate promoter, whereas RAP74 is dispensable eral phosphorylation sites for many protein kinases including for initiation but is required for very early elongation, implying casein kinase 11, protein kinase C, and protein kinase A (40, that RAP74 functions in promoter escape by RNA polymerase 59).' These sequences might represent possible phosphorylaI1 only after transcription is initiated. Their experimentswere tion sites i n vivo, some of which might be responsible for the performed in TFIIF-depleted crude nuclear extracts which con- regulated activity of TFIIF. However, the identification of such tain factors affecting the initiation or elongation of transcrip- residue(s) or protein kinase(s) that catalyzes it needs further tion, including SI1(501,SI11 (511,TFIM (81, and possibly other investigation. Finally, our report that TFIIFactivity could be regulated by unidentified ones. Although several studieswere performed to characterize thesefactors (8,50-53), it is not known whether or posttranslational modification, particularly by protein phosnot these factors can function a t a nearly stageof elongation or phorylation, is the first report on the regulation of the activity interact with TFIIF to affect its function. RAP30 contains a of general transcription initiation factors of mammalian RNA sequence homologous to region l b and 2 of the sigma factors of polymerase 11. The present study might shed light on other bacteria (54), the putative binding domain to RNA polymerase regulation mechanisms of TFIIF action in the complicated seto sufficient for the recruitmentof the ries of protein-protein interactions on both the unregulated I1 (55),and is reported be of transcription. enzyme to the DAB complex (19). Furthermore, it has been basal and the regulated initiation shown recently that RAP30 directly interacts with the amino thank Dr. S. M. Weissman for critical reading terminus of TFIIB (56). Thus, RAP30 might be the likely sub- of Acknowledgments-We the manuscript and Drs. A. J. BerkandD.Reinbergfor their unit of TFIIF that interactsdirectly with the component(s) of generous giRs of the plasmid pETHIID and phIIB, respectively. We the DBPolF complex. However, these results, including those also thank Dr. R. G. Roeder for the kind gift of the plasmid pMLC2AT. by Chang et al. (49), do not necessarily exclude a modulatory function of RAP74 in initiation. Our results strongly indicate REFERENCES that RAP74 is also involved in preinitiationcomplex formation A. G., and Weinmann, R. (1989) FASEB J. 3, 1723-1733 1. Saltzman, by modulating its efficiency. In fact, evidence has been pre2. Sawadogo, M., and Sentenac, A. (1990) Annu. Reu. 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