Danny Reinberg$ and Robert G. Roeder. From the ... Present address: Dept. of Biochemistry, Rob- ...... Dynan, W. S., and Tjian, R. (1985) Nature 316, 774-778.
Vol. 262, No. 7, Issue of March 5, pp. 3331-3337, 1987 Printed in U.S.A.
THEJ O U R N A L OF BIOLOGICAL CHEMISTRY 1987 hy The American Society of Biological Chemists, Inc.
K
Factors Involvedin Specific Transcription by Mammalian RNA Polymerase I1 TRANSCRIPTIONFACTORIISSTIMULATESELONGATIONOF
RNACHAINS* (Received forpublication, August 29, 1986)
Danny Reinberg$ and Robert G . Roeder From the Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021
A factor that stimulates random transcription of purified DNAs by RNA polymerase I1 has been partially purified and analyzed with respect to its possible role in specific transcription from class I1 promoters. Studies of the effect of this factor (transcription factor 11s) on transcription from the adenovirus major late promoter in a systemreconstituted with RNA polymerase I1 and purified factors (IIA, IIB, IIE, and IID) indicated that it acted subsequent to the initiation step and that it stimulated the rate of elongation. Kinetic experiments indicated that the factor affected the efficiency with which the RNA polymerase I1 passed through pausing sites. The relationship of transcription factor 11s to a protein previously purified from Erlich ascites tumor cells (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588) was also studied.
RNApolymerase I1 is acomplex multisubunit enzyme responsible for the synthesisof hnRNA (1).This enzyme thus plays a vital role in the process of gene expression and has been the subject of considerable research (for review, see Ref. 2). Nonetheless, it has been difficult to obtain detailed information about many aspects of the catalytic reaction, especially in promoter-specific transcription reactions. This results in part from the fact that purified RNA polymerase I1 cannot by itself begin specific transcription (initiation at the i n uiuo CAP site)from promoters i n uitro, but must be supplemented by other components present in crude extracts (3-5). In the case of human HeLa cells, transcription from the adenovirus major late promoter (MLP)’by RNA polymerase I1 has been shown to require a minimum of four separable factors (IIA, -B, -E, and -D)(6-11) which have been shown tobe involved in the formationof a preinitiation complex (10-11). Given the complexity of natural templates, it is reasonable to assume that the elongation of an RNA chain may be a highly controlled process and that the elongation and/or termination efficiency might be affected by accessory pro-
* This work was supported by National Institutes of Health Grants CA 34223, CA 34891, and CA 18213 (to R. G. R.) and with general support from the Pew Trust to The Rockefeller University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact. j Recipient of a fellowshipfrom the DammonRunyon Walter Winchell Cancer Fund.Present address: Dept.of Biochemistry, Robert Wood Johnson Medical School, UMDNJ at Rutgers University, 675 Hoes Lane, Piscataway, NJ 08854-5635. ‘The abbreviations used are:MLP, major latepromoter; Ad2, adenovirus 2; TF, transcription factor.
teins. Several studieshave indicated the presence of proteins able to stimulate RNApolymerase I1 in a random transcription reaction (with initiation at nicks, gaps, or ends of the DNA molecule) (12-15). Natoriand co-workers (16) have purified such an activity from Ehrlich ascites tumor cells (SII) and showed that it interacted with RNA polymerase I1 (17). Whereasthey also foundthatantibodiesagainstthe SI1 protein inhibited specific transcription both inisolated nuclei (18)and in a crude reconstituted system that containedAd2MLP (19), the exact role of this factor in the overall transcription reaction has remained unclear. It is becoming clear that a number of proteins can interact with RNA polymerase 11. We have presented evidence that a transcription factor required for specific initiation of transcription (TFIIE) specifically interacted with RNA polymerase I1 (10). Furthermore, Sopta et al. (20) used affinity chromatography with columns containing immobilized calf thymus RNA polymerase I1 to isolate three proteins (Rap30,38, and 72) that interacted with the enzyme. In addition, Kane and Chamberlin (21) have purified a HeLa cell activity, “renaturase,” that dispalces the nascent RNA chain from the template and that could only be detected if the factor was added at thebeginning of the reaction (with RNA polymerase I1 and DNA); this suggested an interaction between renaturase and RNApolymerase 11. In our efforts to learn about mechanism the by which RNA polymerase I1 catalyzed chain elongation in the promoterspecific transcription reaction, we have purified from HeLa cells an activity (transcription factor 11s) that specifically stimulatedthe nonspecific transcription of purified DNA templates by RNA polymerase 11. The role that this protein plays in a specific transcription reaction reconstituted with purified transcriptionfactorshas beenstudied.Our data indicate thatTFIIS acted after the initiation step and that it affected the efficiency of the elongation reaction. The relationship of the TFIIS protein to the proteinpreviously purified from Ehrlich ascites tumor cells (SII) was also studied. MATERIALS ANDMETHODS
Chemicals-Nucleoside triphosphates werefromPharmacia P-L Biochemicals. Adenosine 5”triphosphate purified by high performance liquidchromatographywasfromICN.[cI-~’P]GTP and [3H] UTP werefrom New England Nuclear. Heparin was fromSigma. Polyacrylamide and bisacrylamide were from Bio-Rad. Ammonium sulfate (ultrapure) was from Schwarz/Mann. All other reagents were from Sigma orFisher. Resins-DEAE-cellulose (DE52) andphosphocellulose (P-11)were from Whatman. Heparin-Ultrogel was fromLKB. Single-stranded DNA-agarose was from Bethesda Research Laboratories. Enzymes-RNase-free DNase, terminal deoxynucleotidyltransferase,andrestrictionendonucleases werefromBethesdaResearch Laboratories. DNAs-High molecular weight calf thymus DNA was from Phar-
3331
3332
Purification of RNA Polymerase 11Factors
macia P-L Biochemicals. Plasmid pD139DNA (10)and plasmid pSmaF DNA (3), both containing Ad2-MLP, were prepared by the alkali lysis method as previously described (22). Assay for RNA Polymerase 11 (Nonspecific Transcription Reaction)-This reaction measured the activityof RNA polymerase I1 on random DNA templates in which transcription initiation preferentially started at nicks, gaps, or ends of the DNA molecule. Reactions were performed in a total volume of 25 p1 and contained40 mM TrisHCl, p H 7.9, 2 mM MnC12,2 mM dithiothreitol, 70 mM ammonium sulfate, 5% (v/v) glycerol, 0.6 mM each ATP, CTP, and UTP,50 p M [c~-~'P]GTP with a specific activity of 500 cpm/pmol, and either 25 pg/ml 4x174 DNA or 100 pg/ml calf thymus DNA. Reactions were incubated a t 37 "C for 20 min. The amountof radiolabeled nucleotides incorporated into RNA was measured on DE81 paper as previously described (23). Assay for RNA Polymerase II Stimulatory Activity-This assay measured the stimulation of RNA polymerase I1 activity on nonspecific DNA templates in the presence of TFIIS protein. The reaction was carried out under the conditions described for RNA polymerase I1 but also contained 20 mM KCl. The amounts of RNA polymerase I1 and TFIIS added are indicated in thefigure legends. Assay for Specific Transcription Reaction-The conditions used to study initiation of transcription from Ad2-MLP were as previously described (10) and contained, as described in the figure legends, the purified transcription factors IIA, -B, -E, -D and RNA polymerase 11. The different proteins were purified as previously described (10, 11). Products of reactions tobe analyzed onpolyacrylamide-urea gel were treated asdescribed (10). Preparation of Poly(dC)-tailed Template-Plasmid pD139DNA (10) was linearized with the restrictionendonuclease PstI. Two DNA fragments were produced, one of 4.3 X lo3 base pairs and the other of 5.1 X lo3 base pairs. The terminal addition reaction of poly(dC) was carried out using the mixture of both DNA under the conditions described by Kadesch and Chamberlin (24). Purification of TFIIS-HeLa cell nuclear extracts (7) were fractionated on a phosphocellulose column; and the 0.5 M KC1 eluates, which contained factors IIB, -E, and-S, were further fractionated on aDEAE-cellulose column as detailed elsewhere (10).TheTFIIS activity was present (along with TFIIB) in the 0.1 M KC1 DEAEcellulose flow-through fraction. For further purification of TFIIS, the combined flow-through fractions (136.4 mg of protein, 230 ml) were pooled and loaded at a flow rate of 90 ml/h onto a single-stranded DNA-agarose column (3 mg of protein/ml of resin) equilibrated with Buffer C (20 mM Tris-HC1, pH 7.9 (at 4 "C), 20% (v/v) glycerol, 0.2 mM EDTA, 10 mM P-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride) containing0.1 M KC1. The flow-through fractionscontaining TFIIS activity were pooled and dialyzed for 12 h against 200 volumes of Buffer A (20 mM Tris-HC1, pH 7.0, 20% (v/v) glycerol, 0.2 mM EDTA, 10 mM @-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride) containing 0.1 M KC1. The dialyzed sample (86.4 mg of protein, 275 ml) was loaded a t a flow rate of 50 ml/h onto a heparinUltrogel column equilibrated with Buffer A containing 0.1 M KC1. The activity was eluted from the column with a 10-column volume linear gradient of KC1 (0.1-0.7 M ) in Buffer A. The active fractions were pooled and dialyzed against 200 volumes of Buffer A until the salt concentration of the protein mixture reached 0.1 M KCl. The sample (8 mg of protein, 26 ml) was then loaded a t a flow rate of 3 ml/h onto a phosphocellulose column (10 mg of protein/ml of resin) equilibrated with Buffer A containing 0.1 M KC1. The activity was eluted from the column with a linear gradient (10 column volumes) of KC1 (0.1-0.7 M ) in Buffer A. The active fractionswere pooled, and aliquots of 50 pl were stored a t -80 "C. This procedure yielded a protein with aspecific activity of 1,157,894 units/mg of protein (Table I). Glycerol Gradient Analysis-The interaction of RNA polymerases I1 and 111 with TFIIS was analyzed by glycerol gradient sedimentation. TFIIS, RNA polymerases I1 and 111, and their mixtures were incubated at 4 "C for10 min and thenat 37 "C for 2 min; loaded onto a 5-ml linear gradient of 12.5-25% glycerol in a buffer containing 50 mM Tris-HC1, pH 7.9, 0.1 mM EDTA, 10 mM ammonium sulfate, and 3 mM dithiothreitol; andcentrifuged a t 50 X lo3 rpm for 4.5 h a t 4 "c in an SW 55 rotor. 0.15-ml fractions were collected from the bottom of the tubes, and aliquots (10 p l ) from each fraction were assayed for RNA polymerases I1 and I11 and TFIIS proteinas described above.
RESULTS
Purification and Properties of Actiuity That StimulatesRNA Polymerase 11-Natori and co-workers (16)have isolated from Ehrlich ascites tumor cells a 38,000-Da protein (designated SII) that stimulates RNA polymerase I1 in a random transcription reaction. In our efforts to understand the mechanisms by which RNApolymerase I1 catalyzedelongation, especially inpromoter-specific transcription reactions, we havesimilarlypurifiedfrom HeLa cell nuclear extracts a protein(TFIIS)thatstimulated RNApolymerase I1 ina random transcription reaction. The assay used to purify the protein was a modification of the one previously reported (16) and measured the stimulation of a partially purified HeLa RNA polymerase I1 when a double-stranded DNA was used to direct transcription (see "Materials and Methods"). The protein was purified as described in the legend to TableI and under"Materialsand Methods." High-salt gel filtration through Sephacryl AcA44 suggested an apparent molecular size of about 40,000 Da (data not shown). In the nonspecific transcription reaction (random initiation),TFIIS affected boththerateandthe yield of the reaction (Fig. 1). The maximal extent of TFIIS-catalyzed stimulation of RNA synthesis was not affected by the concenTABLEI Purification of TFIIS Protein
Units x 10-6"
Specific activity unitslmg
protein
mg
Nuclear extract 3,724 219.8 Phosphocellulose 27.5136.4 201,612 DEAE-cellulose 457,175 Single-stranded DNA-agarose 39.5 86.4 8 6.0 750,000 Heparin-Ultrogel 2.2 1.9 1,157,894 Phosphocellulose One unit is defined as the amount of protein that stimulates the activity of RNA polymerase I1 in 1unit under the conditions described under "Materials andMethods."
10
20
(mln 1
FIG. 1. Time course of transcription reaction carried out in the presence and absence of TFIIS. Reaction conditions were as described under "Materials and Methods"with the following modifications. Reactions were carried out in a 200-pl volume and received calf thymus DNA (20 pg), RNA polymerase I1 (0.18 pg, 119,700 units/ mg of protein), and TFIIS(phosphocellulose fraction VI, 3 pg) (0)or buffer (0).Reactions were incubated at 37 "C. At each time point indicated, a 25-p1 aliquot was removed, spotted onto a DE81 filter, and processed for counting. The amountof [c~-~'P]GMP incorporated intothe25.~1 aliquot is shown. The uertical bars indicate -fold stimulation produced by TFIIS.
Purification of RNA Polymerase 11 Factors
3333
tration of RNA polymerase used in the reaction but was dependent upon TFIIS protein concentration, and it rarely exceeded 5-fold (seebelow). The proteinpurified fromEhrlich ascites tumorcells was reported to effect a higher stimulation (10-fold) that was both specific for double-stranded DNA and inhibited by concentrations of ammonium sulfatehigher than 0.02 M (25). The protein purified from HeLa cells showed no DNA template specificity since comparable levels of stimulation by TFIIS were observed with double-stranded DNA (calf thymus DNA and Xenopus DNA), poly(dC)-tailed DNA, I 5 IO and single-stranded DNA (4x174 DNA and denatured calf (mm) thymus DNA) (see below). Stimulation of transcription from I I double-stranded DNA by factor IIS was not affected by the DNA XTP'5 IISprolew addition of ammonium sulfate up to70 mM. The stimulation R N A polymerasen Hlwf In observed with single-stranded DNA was optimal at 60 mM FIG. 2. Effect of TFIIS on transcription from heparin-reammonium sulfate.At the moment,we do not understand the sistant complexes. Two 300-pl reaction mixtures that contained 20 basis for the apparentdifferences between the HeLa and the mM Tris, pH 7.5, 2 mM MnC12, calf thymus DNA (25 pg), and RNA Ehrlichascitestumor cell factors; however, the degree of polymerase I1 (0.22 pg, 119,700 units/mg of protein) were incubated stimulation observed with the human factor varied with dif- at 37 "C. After 3 min of incubation, the nucleoside triphosphates were ferent batches of double-stranded DNA and with the prepa- added, andthe reaction was further incubated for 1 min. Then, heparin (16pg/ml) was added. After 1.5 min, TFIIS (phosphocellulose ration of RNA polymerase I1 used. Thus, it is possible that fraction VI, 4.0 pg) (0)or buffer (0)was added, and thereaction was these differences lie in the particularRNA polymerase I1 and further incubated for different periods of time. At each time point, DNA preparations used rather than in the stimulatory pro- an aliquot of 12.5 plwas removed, spottedonDE81filters, and processed for counting. 15s later, another12.5-p1aliquot was removed teins. TFIIS Affects Elongation of R N A Chains by R N A Polym- andtransferred to atube that contained 90 p1 of 0.1 M sodium erase 11-Previous studies reported that the RNA polymerase pryophosphate and 100 p1 of pheno1:chloroform (1:l); thesample was processed and analyzed by electrophoreses as described under "MaI1 stimulatory activity isolated from Ehrlich ascites tumor terials and Methods." Each point shown represents the amount of cells affected the size of the RNA molecules formed (12) and [ C Y - ~ ~ P I Gincorporated MP into a 25-pl aliquot on the DE81filter stimulated the frequency of initiation by RNA polymerase I1 assay. The vertical bar represents -fold stimulation produced by (26). In order to determine which stage (initiation, elongation, TFIIS. Addition of heparin at thebeginning of the incubation resulted or reutilization of RNA polymerase 11)of the transcription in no reaction (datanot shown). The time scale on the abscissa reaction was affected by HeLa TFIIS, heparinwas used as an represent minutes after theaddition of triphosphates to thereaction. inhibitor of initiation of RNA synthesis.Previousstudies have shown that heparin is a very effective inhibitor of free (unbound) RNA polymerases (27). Since only those polymerase molecules that arein transcription complexes or in tight initiation complexes will be resistant to heparin,a maximum of one round of transcription should takeplace. In the experiment of Fig. 2 (see protocol a t bottom of figure), RNA polymerase I1 was first incubated with double-stranded calf thymus DNA at 37 "C for 3 min to allow the polymerase to bind to the DNA. Next, the ribonucleoside triphosphates were added, and the reaction was incubated for 1min toallow initiation and the formation of elongation complexes. Heparin (12 pg/ml) was then added, and 1.5 min later, either TFIIS or buffer. As shown in Fig. 2,RNA synthesis was stimulated (min in the presence (closed circles) but not in the absence (open circles) of TFIIS. Product analysis after DNase I treatment ON* n.pm indicated that the transcripts formed in the presence of the RNA TI IIS PrOleln TFIIS protein were larger than those made in its absence XTP't (data not shown; see below). The above results, while sugFIG.3. Effect of TFIIS in initiation reaction. Reaction congesting that TFIIS was an elongation factor, did not rule out ditions were as described in the legend to Fig. 2, but the addition of the possibility that the factorcould also stimulate the initia- TFIIS protein (0)or buffer (0)was at thebeginning of the reaction. tion reaction (binding of RNA polymerase to theDNA or the frequency of initiation). If so, then it would be expected that and/or processivity of RNA polymerase 11). the amount of stimulation should be greater when added As indicated above, previous studies of SI1 from Ehrlich duringtheinitiation reaction(before heparin)than when ascites tumor cells were interpreted in terms of a stimulation added afterheparin(when only the elongationreaction is of the frequency of reinitiation (26). However, this result is allowed). To address this question, theprotocol of Fig. 2 was not incompatible with a direct effect of TFIIS only on elonaltered by adding TFIIS or buffer with the polymerase. As gation. Thus, if it were necessaryfor the polymerase to shown in Fig. 3, TFIIS also stimulated transcription under transcribe a fixed length of DNA prior to dissociation and these conditions (open circles),but the extent of stimulation reinitiation, then an elongation factor which permitted a n was identical to that observed in Fig, 2 when the factor was initiated RNA polymerase to accomplish thisfaster could added after initiation (during the elongation phase). Thus, have an apparent but indirect effect upon initiation, simply the resultsof Figs. 2 and 3 indicate that TFIIS stimulated notby facilitating cycling of RNA polymerase. the initiation reaction, but rather elongation the reaction (rate TFIIS Can Be Isolated in a Complex with R N A Polymerase
Potym.role 1
Purification of RNA Polymerase II Factors
3334
II-Studies with the RNA polymerase I1 stimulatory activity levels of activity with Mi2+(uersus Mn”) in the nonspecific transcription assay with double-stranded DNA(l),these obisolated from Ehrlich ascites tumor cells indicated that the purified protein interacted with RNA polymerase I1 (15). In servations might have reflected only a lack of sensitivity in the assay. We therefore reinvestigated the effect of magnesupport of thisfinding, a 38,000-Da protein(presumably analogue to SII) in murine erythroleukemia cell extracts was sium uersus manganese on the stimulatory activity using a selectively bound to an immobilized RNA polymeraseI1 and model double-stranded DNA template. shown to stimulate RNA polymerase I1 in a random transcrip- Kadesch and Chamberlin (24) reported that a DNA moletion reaction (20). In order to investigate the possibility that cule witha deoxycytidine tail was an active template for RNA the purified HeLa stimulatory protein bound to RNA polym- polymerase I1 with magnesium as the divalent cation. RNA erase, glycerol gradient experiments were carried out as de- polymerase I1 will initiate de nouo on the deoxycytidine tail (24, 28). In our studies, Mi2+ and Mn2+ were found to be scribed under “Materials and Methods.” If the two compoequally effective when RNA polymerase I1 activity on this nents interacted or if RNA polymerase I1 were modified by as is evident from the GMP incortemplate was measured, the TFIIS protein, the RNA polymerase activity recovered poration values (picomoles) forlanes 3 and 5 in Fig. 5 . TFIISafter centrifugation should be higher than the control RNA polymerase (preincubated and sedimented but in the absence dependent stimulation of RNA polymerase I1 was observed with both magnesium (4.9-fold) (compare lanes 3 and 4 ) and of the TFIIS protein). As shown in Fig. 4, the RNA polymerase I1 that was preincubated with the TFIIS protein ( B ) manganese (3.5-fold) (compare lanes 5 and 6 ) . Although the was 2.5- and 3.2-fold more active than the RNA polymerase .isolated from the control gradients (A) when assayed, with double-stranded calf thymus DNA or 6x174 DNA, respectively. Control experiments showed that TFIIS sedimented in a distinct position a t t h e t o p of the gradient (Fig. 4C) in the absence of RNApolymerase I1 and that preincubation of TFIIS with RNA polymerase 111 ( D ) had no effect on the activity of this enzyme (compare D and E). Furthermore, when incubated and sedimented with RNA polymerase 111, the TFIIS act,ivity remained at the topof the gradient (data not shown). These results indicated that interactionor modification by TFIIS was specific for RNA polymerase11. Effect of T F I I S o n RNA Polymerase I I Using a Pol.y(dC)tailed Template DNA-A major goal of this study has been to underst.and the role of factor IIS in the specific transcription reaction. In contrast to the observation that this reaction is dependent upon Mi‘+ and is inhibited by Mn2+ (3), studies with the RNA polymerase I1 stimulatory activity isolated from Ehrlich ascites tumor cells (25) or murine erythroleukemia cells (20) suggested that it required manganese for activity. However. because RNA polymerase I1 shows much reduced b3‘P)GMP
Incorporated(pmo1)
Bottom
IO
20 TOP FRACTION
FIG.4. Glycerol gradient analysis of interaction of TFIIS with RNA polymerases I1 and 111. TFIIS (phosphocellulose fraction VI, 45 pg), RNA polymerase I1 (30 pg, 119,700 units/mg of protein), RNA polymerase 111 (75 pg, 40 DNA units/mg of protein), and their mixtures were treated as described in the text and subjected to plycerol gradient, centrifugation as described under “Material and Methods.” Fractions of 150 pl were collected from the bottom ofthe tubes, and 10- or 5-pl aliquots were removed and assayed for RNA polymerase ( A , H , 11, and E ) or TFIIS (C) activity as described under “Materials and Methods” using either calf thymus DNA or 4x174 DNA, respectively.
20
23
II
113
0.9
4.7
3.0 FIG. 5. Effect of TFIIS on RNA polymerase I1 using poly(dC)-tailed DNA. Reaction mixtures (25 pl) were incubated at 37 “C for 20 min as described under “Materials and Methods” with the following modifications: tubes 1 and 2 received calf thymus DNA (2.0 p g ) and manganese ( 2 mM); tubes 3-7 received poly(dC)-tailed PstI-cut pD139 DNA (0.2 pg); whereas tubes 8 and 9 received PstIcut pD139 DNA (carried through the terminal addition reaction, hut incubated in the absence of nucleotides). In addition, tubes 3, 4, 8, and 9 received magnesium (6 mM), whereas tubes 5-7 received manganese (2 mM). All other reagents were as described under “Materials and Methods.” Tubes 2, 4, 6, 7, and 9 received TFIIS (0.35 pg). All tubes hut 7 received RNA polymerase I1 (70 ng, 119,700 units/mg of protein). After incubation, an aliquot of 12.5 pi was removedfrom each tube, spotted on DE81 paper, and processed as described. The other half of the reaction received 75 p1 of 0.1 M sodium pyrophosphate and 100 p1ofphenol:chloroform (1:l).The aqueous phase was removed, brought to 0.3 M sodium acetate, pH 5.5, and precipitated with ethanol. The precipitated material was resuspended in 50 pl with 50 mM Tris-HCI, pH 8.0, 5 mM MgCI,. RNase-free DNase (2 p g ) was added, and the mixture was incubated at 37 “C for 30 min. Samples were extracted with phenol:chloroform, and the aqueous phase was ethanol-precipitated. Samples were resuspended in 20 pl with 10 mM methyl mercury in water, and the products of the above reactions were separated on a 1.2% agarose gel containing 5 mM methyl mercury. The gel was dried on Whatman DE81 paper. The picomoles of (W”~P]GMP incorporated into the complete reaction are shown at the bottom. The bracket indicates the region of full-length (end-toend) RNA transcripts. 43
71
Purification of RNA Polymerase 11 Factors
3335
Mg”-containing reactions that were carried out in the absence of the TFIIS protein produced some full-size products (Iane 31, the presence of the TFIIS protein in the reaction greatly stimulated the productionof this full-size RNA (lane 4 ) . This reaction was absolutely dependent on the presence of deoxycytidine residues on the DNA fragment(lanes 8 and 9 ) . In the Mn”-containing reaction with the deoxycytidinetailed template, TFIISalso stimulated t,he productionof fullsize RNA molecules (lanes *5 and 6 ) . When calf thymus DNA was used as template under comparable conditions, TFIIS stimulatedtranscription 3.9-fold inthepresence of Mn” (Ianps 1 and 2). In this case, however, the transcripts synthesized in response to TFIIS were,as expected, more heterogenous in size (lane 2); whereas no pr0duct.s were observed on t h e gel when TFIIS was omitted from the reaction(lane 1).It is probable that the RNA molecules formed in the latter case were short and that they ran out ofthe gel (lane 1). Reactions that contained the poly(dC)-tailed templates produced RNA molecules of the expected sizes (4300 and 5100 base pairs), 221 although they were present in broad bands. At the present moment, we do not know t.he reasons for the heterogeneity, although it could be that HeLa RNA polymerase I1 can start transcription at any place on the deoxycytidylic tail (average c size of 20-30 nucleotides, assuming that every end of the input 154 DNA was active in the terminal addition reaction). The results presented here indicate that TFIIS protein is (Q-32P)GMP o.4 o.9 active with both magnesium and manganese, thus facilitating I ncoporated 0.6 0.8 analysis in the specific transcription reaction (below), and ( pmol) is agoodtemplatetomeasure thatpoly(dC)-tailedDNA FIG.6. Effect of TFIIS in specific transcription reaction. A, TFIIS stimulatory activity. reaction conditions ( 3 0 pl) were as described under “Materials and E f f d o f TFIIS Protein in Specific Transcription ReactionMethods”andcontainedSmal-cutpSmaF DNA (0.5 fig), TFIIR Previous studies indicated that specific transcription in iso- (single-stranded DNA-agarosefraction, 0.7 p g ) , TFIIE (Sephacryl latednuclei and in a system reconstituted with a purified AcA44, 0.5 p g ) , TFIID (DEAE-Sephacel fraction, 0.28 p g ) , TFIIA (DEAE-Sephacel fraction, 0.8 p g ) , TFIIC(single-stranded DNAadenovirus MLP-containing DNA was inhibited by antibodies agarose fraction, 0.12 p g ) , and RNA polymerase I1 (70 ng, 119,700 to the SI1 protein (18, 19). We have previously shown that of protein). After 20 min at 30 “C, an aliquot of 15 pl was our reconstituted system requiresat least four factors (TFIIA, units/mg removed, spotted on DE81 fitlers, and processed as described. The -R, -E, and -D) in addition to RNA polymerase I1 (6, 10, 11) other half of the reaction was processed as described (10). Products for specific transcription from promoters containing TATA of the reaction were separated by electrophoresis on a 4% polyacrylsequences. When purified proteins (see legendt,o Fig. 6) were amide, 7 M urea gel. R, reactions were as described forA. The reaction used to transcribe the Ad2 major late promoter, we were able that was carried out in the presence of TFIIS received 0.3 pg of the to observe only a modest (2-fold) stimulation by TFIIS when phosphocellulosefraction VI. The products of the reactions were separated by electrophoresis on a 6% polyacrylamide, i M urea gel. the products of the reaction were analyzed by measuring the amount of [w:”PJGMPincorporated(Fig. 6A). However, After 45 min at 30 “C, the ribonucleoside triphosphates were when the discrete run-off products of the reaction (resulting added and the reaction was incubated for an additional minfromspecificinitiationandelongationtot.heend of t h e ute, at which time heparin was addedto block further initia49; polyac- tion. Triplicate sets of reactions containing either no TFIIS, template) were analyzed by electrophoresis in a rylamide-urea gel, theTFIIS-dependentstimulationwas 0 min, or TFIISadded at 47minwere TFIISaddedat greater (about G-fold, compare lanes 1 and 4 in Fig. 6 A ) . A incubated at 30 “C for various times, and the productsof the likely explanation is that elongation took place at a much 65 reactionswereanalyzedbyelectrophoresisthrougha slower rate in the absence of TFIIS and that small RNA polyacrylamide-urea gel. As shown in Fig. 7, the production molecules(correctlyinitiatedbutincompletelyelongated) of full-size RNA molecules (arrow),first noticeable at about were selectively lost during the electrophoretic analysis. To 10 min, was stimulated by the presence of TFIIS, in agreement investigate this possibility, the productsof the transcription with previous results on the general stimulatory activity of reactions performed in the presence and absence of t h e T F I I S the protein. Moreover, TFIIS was equally effective whether protein were analyzed by electrophoresis through a 6% poly- added before (lanes 11, 14, and 17) or after (lanes 12, 15, and acrylamide-urea gel (Fig. 6R). Under these conditions, small 18) heparin, indicating that i t acted after initiation, again in RNA molecules (see arrows) could be identified as products agreement with our previous results (10). Furthermore, reacin t.he reaction that lacked TFIIS (lane 2), whereas a greater tions that did not contain the TFIIS produced, at early times, proportion of the label was in completely elongated products two small RNA molecules of approximately 120 (band b) and (536 nucleotides) in the reaction wit,h T F I I S (lane 1). 175 (hand a ) nucleotides (lanes 1, 4, 7, 10, 13, and 16). T h e T o investigate further this observation, we analyzed the 120-nucleotide RNA appeared to be chased to the 175-nucleoproducts of the specific transcription reaction in the presence tideRNA.The175-nucleotideRNAwasalsoobserved at and absenceof the TFIIS protein when elongation was limited early times in reactions that contained the TFIIS protein to onlyoneround(seeprotocolin Fig. 7). Transcription (lanes 2 and 3 ) ; but in this case, the RNA was apparently complexes were formeda t t h eAd2 major late promoter in the chased to full-size products (lanes 11, 12, 14, 1.5, 17, and 18). presence and absence of TFIIS as previously described (10). Reactions carried out in the absence of TFIIS seemed to IC
Purification of RNA Polymerase I I Factors
3:m 47
0 I
46
45
DNA IIB,IIE,nD,lIA RNA polymerase +IIS
XTP's Heparin
IIS Buffer
a
396 344 298 517
mapped in the major late transcription unit of adenovirus serotype2,both in uiuo andinisolatednuclei(29). The importance of this correlation is presently unknown, but it may indicate that TFIIS acts as an anti-terminator or that the two RNA molecules detected by Maderious and ChenKiang (29) maybeduetoinefficiencyintheelongation reaction. Preliminary results suggest that the site that lies 175 nucleotides downstream from the CAP site is a strong pausing site; thus, the addition of TFIIS after 10 or 15 min of elongation (under the conditions described in the legend to Fig. 7 ) resulted in t,he conversion of about 50% of band a to full-size products.2 These results, while preliminary, suggest that under thein oitro conditions used in Fig. 7, t.his site was a stop site at which the RNA polymerase paused and remained bound to the template. If these sites were to be used as a termination, as suggested by Maderious and Chen-Kiang (29), the above results suggest that the termination reaction (release of the RNA molecule and of RNA polymerase 11) may require an additional factor that was absent in our reconstituted system. DISCUSSION
We have previously shown that specific initiation of tran221 a scription at the adenovirus major late promoter required minimum of four HeLa cell transcription factors (HA,-R, -E, I1 (6,8, 10, 11). Here, and -D) in addition to RNA polymerase 0we have purified a HeLa cell activity (TFIIS) that specifically I1 in a randomtranscription stimulatedRNApolymerase 154 reaction, and we have analyzed its role in the specific transcription reaction. bUsing the purified reconstituted system mentioned above, we have shown that the addition of TFIIS had an effect in the overall level of specific transcription from the major late promoter in a template generating a fully elongated product of 536 nucleotides. Furthermore, when the reaction was studied under conditions which restricted initiation to one round (the presence of heparin), it was possible to demonstrate that e TFIIShadnoeffectintheinitiationreactionbutrather stimulated the postinitiation phaseof the transcription reac75 tion. The studies presented here (those of Fig. 7 ) suggest that I1 TFIIS affected the efficiency with which RNA polymerase passed through discrete pausing sites on the DNA template FIG. 7. Effect of TFIIS in specific transcription reaction and thatit therefore affected the overall rate of the elongation when elongation was limited to one round. Reaction mixtures reaction. These results are in agreement with those on the in 210 pl contained 20 mM Tris-HCI, pH 7.9, 8 mM M&h, 10 mM ammonium sulfate,100 p~ high performance liquid chromatography- effect of the factor in the general nonspecific transcription purified ATP, SrnaI-cut pSmaFDNA (3.0p g ) , TFIIR (single-stranded reaction; these studies (data of Figs. 2 and 3) indicate that TFIIS affected the elongation reaction. DNA-agarose fraction, 4.9 p g ) , TFIIE (Sephacryl AcA44 fraction, 4 p g ) , TFIlD (DEAE-Sephacel fraction, 2 pg), TFIIA (DEAE-Sephacel Previous studies have identified a murine factor (SII) that fraction, 5.6 p g ) , TFIIC (single-stranded DNA-agarose fraction, 0.72 interactedwith(17)andspecificallystimulated (16) RNA p g ) , and RNA polymerase I1 (0.42 pg, 119,700 units/mg of protein); polymerase I1 in a randomtranscriptionreaction. At the to one of three tuhes, TFIIS (phosphocellulose fraction VI, 4.2 p g ) present moment, it is unknown if the murine (Ehrlich ascites was added, and the reaction was incubated a t 30 "C for 45 min. At this time, the ribonucleoside triphosphates were added; and after 1 tumorcell)andhuman(HeLacell)factorsarethesame. However, even though we have observed some differences in min a t 30 "C, heparin (I6 pg/ml) was added. After another minute of incuhation, TFIIS or buffer was added. Reactions were incubated for the nonspecific transcription reaction (those presented in the different periods of time as indicated. At each time point, aliquotsof text) between the two factors, other results suggest that they 25 pl were removed and transferred to tuhes containing stop mixture might he the same. First, both activities were specific for RNA (75 pl; see Ref. 10) and pheno1:chloforom ( 1 : l ) (100 pl). Samples were processed as described under "Materials and Methods" under "Prod- polymerase 11. Thus, the murine factor stimulated the homologous RNA polymerase11, but not the homologous polymerase uct Analysis" (in Ref. 10). The productsof the reaction were separated I (12): whereas the HeLa factor stimulated RNA polymerase hy electrophoresis on a 6% polyacrylamide, 7 M urea gel. I1 from different sources, but. failed to stimulate RNA polym111 or Escherichia coli RNA polymerase.:' Second, both erase accumulate this 175-nucleotide RNA, although a small perproteins appear to have a similar molecular size. T h e SI1 is pass through this site and centage was apparently able to 38,000 Da, and it appears as a doubletinsodiumdodecyl become full-size products after 10-20 min of incubation (lanes sulfate-polyacrylamide gel (15,16):whereas TFIIS activity IO. I.']. and 16). Interestingly, the 120- and 175-nucleotide RNAs map at or ? D. Reinherg, unpuhlished ohsewation. close t o the sites in which premature termination has been :' D. Reinherg and R. G. Roeder, unpuhlished ohservation.
.
I
.
-
.
Purification of RNA Polymerase 11Factors analyzed here was found to have a molecular size of 38,0001. 42,000 Da when analyzed on sodium dodecyl sulfate-polyacrylamide geL3 Third, both factorswere also shown to interact probable with thepurified RNA polymerase 11. Thus, it seems 2. that the minordifferences observed in the catalytic reactions 3. lie in the RNA polymerase I1 and DNA preparations used 4. rather than in the stimulatory proteins. Studies using antibodies against thepurified SI1 protein in a HeLa crude transcription system or in isolated nuclei sug5. gested that antigens cross-reacting with the antibodies were 6. essential for accurate transcription (18,19). However, because 7. thosestudies were carriedoutincrudesystemsin which reinitiation of transcription was not blocked, it was not pos8. sible to deduce the step, during the specific transcription reaction, a t which the factor was required. Moreover, because 9. SI1 protein interacted with RNA polymerase I1 and was part of a transcription complex (17), these studies did not rule out 10. the possibility that the inhibition observed withthe antibodies 11. was an indirect effect due to coprecipitation of RNA polymerase 11. The results presentedin this paper indicate that the 12. 13. HeLa RNA polymerase I1 activity acted after the initiation step, both in specific and nonspecific transcription reaction, and was not an essential componentof the RNA polymerase 14. I1 transcription machinery in vitro; however, we cannot rule 15. out the possibility that trace amounts of the factor contaminated RNA polymerase 11. Other studies have shown that this16. factor is distinct from the known transcriptioninitiation 17. factors for the major late promoter (10). The in vivo role of the protein is unknown at present andwill only beunderstood 18. with more detailed investigationof the initiation, elongation, 19. and terminationreactions. Many steps of the transcription reaction can affect the 20. efficiency with which a given gene is transcribed. It possible is that most of the regulation is executed at the initiation level 21. (including preinitiation), although secondary levels of regulation may well take place during elongation. The state of 22. phosphorylation of RNA polymerase I1 or of the transcription factors may be a suitable step for secondary regulation. It is 23. relevant to note that SI1 protein can exist in both the phosphorylated and dephosphorylated stage (15,30). Furthermore,24. Weinmann and co-workers have shown that 5,6-dichloro-125. /%D-ribofuranosylbenzimidazole concentrations which inhibitedthe specific in vitro transcription reaction (31) were similar to those which inhibited a casein kinase type I1 (32). 26. They proposed that this kinasemay act directly or via one of 27. the transcription factors (possible 11s) in the transcription reaction ( 3 3 ) . A further analysis with in vitro transcription reaction reconstitutedwith morepurified proteins should 28. facilitate an analysis of the mechanismof action of the various 29. initiation and elongation factors. Acknowledgments-We wish to thank Dr. Elizabeth Slattery, Dr. K. J. Marians, Dr. Lynne Vales, Dr. R. Weinmann, Dr. J. Rappapport, and Dr. M. Horikoshi for helpful discussion while the work was in progress. We also thank Dr. Jeff Field for helping us with the preparation of the poly(dC)-tailed template and Drs. K. J. Marians, N. Heintz, B. Jack, andLynne Vales for comments onthe manuscript. The technical assistance of M. E. Perry in preparing the nuclear extract is also acknowledged.
30. 31. 32. 33.
3337 REFERENCES
Roeder, R. G . (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds) pp. 285-330, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Sentenec, A. (1982) CRC Crit. Reu. Biochem. 18,31-91 Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979) Cell 18,469-484 Heintz, N.,and Roeder, R. G . (1982) in Genetic Engineering (Setlow, J. K., and Hollarnder, S., eds) Vol. 4, pp. 57-82, Plenum Press, New York Dynan, W. S., and Tjian, R. (1985) Nature 3 1 6 , 774-778 Matsui, T., Segell, J., Weil, P. A., and Roeder, R. G . (1980) J. Biol. Chem. 2 5 5 , 11992-11996 Dignam, D. J., Lebovitz, R. M., and Roeder, R. G . (1983) Nucleic Aczds Res. 1 1 , 1475-1489 Sawadogo, M., and Roeder, R. G. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,4334-4398 Fire, A., Samuels, M., and Sharp, P. A. (1984) J. Biol. Chem. 259,2509-2516 Reinberg, D., and Roeder, R. G . (1987) J. Biol. Chem. 2 6 2 , 3310-3321 Reinberg, D., Horikoshi, M., and Roeder, R. G . (1987) J. Biol. Chem. 262,3322-3330 Natori, S. (1982) Mol. Cell. Bid. 4 6 , 173-187 Barbiroli, B., Monti, M. G., Moruzzi, M. S., and Mezzetti, G . (1977) Biochim. Biophys. Acta 479.69-79 Legraverend, M., andGiazer, R. I. (1980) Biochim. Biophys. Acta 607,92-101 Horikoshi, M., Sekimizu, K., Hirashima, S., Mitsubashi, Y., and Natori, S. (1985) J. Bid. Chem. 2 6 0 , 5739-5744 Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 1 8 , 1582-1588 Horikoshi, M., Sekimizu, K., and Natori, S. (1984) J. Biol. Chem. 259,608-611 Ueno, K., Sekimizu, K., Mizuno, D., and Natori, S. (1979) Nature 277., 145-146 ~~Sekimizu, K., Yokoi, H., and Natori, S. (1982) J . Biol. Chem. 2 5 7 , 2719-2721 Sopta, M., Carthew, R. W., and Greenhlatt, J. (1985) J . Biol. Chem. 2 6 0 , 10353-10360 Kane, C. M., and Chamberlin, M. (1985) Biochemistry 24,22542262 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Schwartz, L. B., Sklar, V. E. F., Jaehning, J. A., Weinmann, R., and Roeder, R. G. (1974) J. Biol. chem. 2 4 9 , 5889-5897 Kadesch, T. R., and Chamberlin, M. J. (1982) J. Bid. Chem. 257,5286-5295 Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 1 3 0 , 6972 Sekimizu, K., Horikoshi, M., and Natori, S. (1984) J . Biochem. (Tokyo) 96, 1859-1865 Chamberlin, M . J. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds) pp. 17-68, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Dedrick, R. L., and Chamberlin, M. J. (1985) Biochemistry 2 4 , 2245-2253 Maderious, A., and Chen-Kiang, S. (1984) Proc. Natl. Acad. Sci. U. S. A . 81,5931-5935 Sekimizu, K., Kubo, Y., Segawa, K., and Natori, S. (1981) Biochemistry 2 0 , 2286-2292 Zandomeni, R., Mittleman, B., Bunick, D., Ackerman, S., and Weinmann, R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,31673170 Zandomeni, R., and Weinmann, R. (1984) J. Biol. Chem. 2 5 9 , 14804-14811 Zandomeni, R., Zandomeni, M. C., Shugar, D., and Weinmann, R. (1986) J . Biol. Chem. 2 6 1 . 3414-3419 ~~~
