T H E ,IOURNAI. OF BIOLOGICAL CHEMISTRY <. 1984 hy The American Society of Biological Chemists, Inc

Vol. 259, No. 8, Issue of April 25, pp. 5321-5326, 1984 Printed in U.S.A.

Energy Requirement for Specific Transcription Initiation by the Human RNA Polymerase I1 System*

(Received for publication, October 4, 1983)

Michele Sawadogo and Robert G . Roeder From The Rockefeller University, New York, New York 10021

The energy requirement for specific transcription initiation and elongation by the human RNA polymer- ase 11 system was studied in vitro using partially pu- rified transcription factors from HeLa cell nuclear extracts. The synthesis of the 536-nucleotide long run- off transcript resulting from initiation at the adenovi- rus major late promoter was found to be dependent upon the presence of either ATP or dATP (with the imido derivative adenyl-5”yl imidodiphosphate being used as the substrate for the RNA polymerase elonga- tion reaction). An identical requirement for hydrolysis of the phosphate bond in an adenosine nucleotide was observed for the synthesis of the decanucleotide tran- scribed from the major late promoter in the absence of the GTP substrate. In contrast, the nonhydrolyzable analog adenyl-5”yl imidodiphosphate fully substitutes for ATP during the subsequent elongation of these short transcripts, which demonstrates that the energy requirement occurs at an earlier step of the transcrip- tion reaction. Thus the particular transcription factor that requires ATP (or dATP) hydrolysis for its function must act prior to, or concomitant with, formation of the first few phosphodiester linkages by the RNA po- lymerase 11.

The transcription of specific genes involves initiation, and presumably termination, by the RNA polymerase molecule at particular sites on the DNA, but the mechanisms involved in these processes in eukaryotic cells are still poorly understood. However, the ability to modify and express purified genes has allowed the identification of particular DNA sequences im- portant as transcription initiation or regulation signals (for reviews, see Refs. 1 and 2). In addition, there have been developed soluble cell-free systems which mediate accurate transcription initiation (3, 4), a first step toward the identi- fication of the cellular components involved in this process.

In the case of genes transcribed by RNA polymerase 11, the initial chromatographic fractionations of soluble extracts have demonstrated a requirement for several protein factors in addition to RNA polymerase (5). One of these factors has been purified to homogeneity but its in uitro requirement appears to be related to its ability to bind to nicks in the DNA template, thereby eliminating random transcription initiation by RNA polymerase I1 (6). Although none of the other factors (5, 7; see also Ref. 8) are yet purified to homogeneity, one or

* This work was supported by Research Grant CA 24223-01 and CA 24891-01 from the National Institutes of Health and by Research Grant NP 284 B from The American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

~~~~~~~~ ~~~

more of these appears to interact with specific DNA sites, resulting in the formation of a preinitiation complex (9).

Recently, Bunick et al. (10) have reported that specific transcription by RNA polymerase I1 in a HeLa whole cell extract is inhibited when the ATP is replaced by AMP-PNP,’ although this nonhydrolyzable analog can be accepted as a substrate by the polymerase. This inhibition was observed with several genes having different initiating nucleotides, but not with other imidonucleotides. These results indicated that ATP hydrolysis is required for specific transcription inde- pendently of the RNA capping process, but did not clearly establish the point a t which the ATP hydrolysis is required.

We are presently purifying from nuclear extracts (11, 12) the different components required to reconstitute specific transcription initiation events in vitro. During the course of this work, we repeatedly observed that a DNA-dependent ATPase activity co-chromatographed with transcription fac- tor TFIIE through all of the many purification steps that were attempted. In contast, the other transcription factors could be purified away from any contaminating ATPase ac- tivities.’ This particular ATPase hydrolyzes dATP as well as ATP, which prompted us to investigate whether dATP could fulfill i n uitro the energy requirement of the specific transcrip- tion. We report here that transcription can take place in the presence of AMP-PNP as an RNA polymerase substrate and dATP as an energy source. This property was further ex- ploited to investigate which of the transcriptional steps (preinitiation, initiation, or chain elongation) requires hy- drolysis of an adenosine triphosphate analog.

MATERIALS AND METHODS

Nucleotides-High pressure liquid chromatography-purified ribo- nucleoside triphosphates were purchased from ICN, AMP-PNP from Boehringer, dATP from Sigma or P-L Biochemicals (HPLC-purified, ribonucleotide-free), and the labeled nucleotides from Amersham.

Enzymes-RNase A was from Sigma and RNase T, from Calbi- ochem. Bacterial alkaline phosphatase was obtained from Bethesda Research Laboratories or New England Nuclear.

Transcription Factors-Nuclear extracts were prepared from HeLa cells as described previously (11) except that all the buffers contained Tris (20 mM, pH 7.3, at 20 “C) instead of Hepes and that Triton X- 100 (Sigma) was added to 0.2% final to the extracts just prior to dialysis. These extracts were purified by chromatography on PI1 and DE52 columns as described (12). To avoid any contamination by endogenous nucleotides, the P11 breakthrough fractions containing TFIIA (44 ml, 132 mg of protein) were applied to a DE52 column (15 ml, 1.7 X 5.6 cm) which was eluted with a 0.033-0.5 M ammonium sulfate gradient. The active fractions were pooled and dialyzed against a buffer without salt until the conductivity was that of the same buffer with 0.1 M KC1. Protein concentrations were determined by the method of Bradford (13).

In Vitro Transcription-Transcription reactions contained 11 mM

‘ The abbreviations used are: AMP-PNP, adenyl-5”yl imidodi- ~~~~~~ ~ ~ ~ ~~

phosphate; Ad2 ML, Adenovirus 2 major late; ML major late. M. Sawadogo, unpublished observations.

5321

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

5322 Specific Transcription Initiation Energy Requirement MgC12, 20 pg/ml of Sma I-cut pSmaf plasmid DNA (3) and the nucleotide concentration indicated in the figure legends. 60% of each reaction volume (25 or 50 pl) was comprised of column fractions, all dialyzed against a buffer with 0.1 M KC1 and 20% glycerol, such that the final salt and glycerol concentrations were 0.06 M and 1276, respectiveLy. Hepes (pH 8.4) was added to a 40 mM final concentration to compensate for the pH change of the Tris buffer with temperature.

The reconstitutions contained, for 25-p1 reactions, the following fractions of the different transcription factors: for TFIIA, DE52 fraction, 2 pl, 1.5 pg of protein; for TFIIB, DE52 0.1 M KC1 break- through fraction, 4 pl, 1.4 pg of protein; for TFIIE, DE52 0.25 M KC1 step, 4 pl, 4.4 pg of protein; finally, for TFIIC and TFIID, P11 1 M step fraction, 5 pl, 5 pg of protein. Since endogenous RNA polymerase I1 was contained in the TFIIE fraction, exogenous enzyme was omitted.

Preparation of RNA Molecular Weight Markers-Run-off tran- scripts from Ad2 ML were synthesized in a 2 0 0 4 reaction (prepared as described above) in the presence of [c~-'~P]CTP as the labeled nucleotide. These transcripts were then purified by phenol and chlo- roform extraction, precipitated with alcohol, resuspended in 10 mM Tris (pH 7.5). 0.2 mM EDTA buffer and digested by RNase TI (40 units) for 30 min at 37 "C. The resulting mixture of oligonucleotides was used as molecular weight markers without any further purifica- tion. The lower RNAs were 16, 12, 11, and 8 nucleotides long, as expected from the adenovirus sequence. The radiochemical degrada- tion of the 16-mer generated, with time, an additional 14-nucleotide long RNA.

RESULTS AND DISCUSSION

ATP or dATP Requirement for Specific Transcription of Ad2 M L in an in Vitro Reconstituted System-We chose to study the ATP requirement for specific transcription using a system reconstituted in vitro with semipurified transcription factors from HeLa cell nuclei because such a reconstitution is totally dependent upon the addition of exogenous nucleotides (see Fig. 1, lane I), and also because the transcriptional activity, as judged by the production of run-off transcripts

A

from the Ad2 ML promoter, is increased severalfold as com- pared to the original extract, presumably because of the removal of inhibitors and/or a greater concentration of active components?

Fig. 1 illustrates the effect of ATP analogs on the produc- tion of run-off transcripts from the Ad2 ML promoter in such a reconstituted transcription system. As originally observed in a HeLa whole cell extract (lo), the replacement of ATP (lane 3) with the nonhydrolyzable analog AMP-PNP (lane 7) almost completely suppressed the specific transcription. The fact that a mixture of ATP and AMP-PNP (lane 5) resulted in a run-off transcript nearly identical in amount to that obtained with ATP alone excludes the possibility of an inhib- itor in the AMP-PNP preparation. Interestingly, when a reaction containing AMP-PNP as a polymerase substrate was supplemented with a low concentration of dATP, the tran- scription was increased 10-15-fold, yielding an amount of specific transcript similar to that obtained in the presence of ATP. Moreover the dATP could not be replaced by AMP or by any of the other three deoxynucleoside triphosphates in allowing the incorporation of AMP-PNP into specific run-off transcripts (Fig. 1B). To exclude artifacts such as an enzy- matic conversion of the dATP (or a degradation product thereof) into ATP, a control experiment was performed in which cu-"'P-labeled dATP was introduced into a similar tran- scription reaction in the absence of any other labeled nucleo- tide (Fig. lB, lane 7). This experiment clearly indicates that the dATP is not incorporated into the RNA. Quantitation of the amount of run-off transcript synthesized in the presence of AMP-PNP and various concentrations of dATP yielded an apparent K , for the dATP of 20 PM (result not shown).

These results imply that when the specific transcription

B

A T P - - + + + + " AMP-PNP - - - - + + + +

d A T P - + - + - + - +

1 2 3 4 5 6 7 0 *a*.

1 2 3 4 5 6 7 "

FIG. 1. Effect of ATP analogs on the production of run-off transcripts from adenovirus major late promoter in an in vitro reconstituted system. RNA was synthesized in the presence of transcription factors purified from a HeLa cell nuclear extract with Sma I-cut pSmaf DNA template as described under "Materials and Methods." The transcription reactions (25 pl each) were incubated for 1 h a t 30 "C, then stopped by the addition of EDTA (10 mM) and sodium dodecyl sulfate (0.2%) and analyzed by electrophoresis on 4.5% acrylamide Tris- acetate-sodium dodecyl sulfate gel. The arrow indicates the 536-nucleotide run-off transcript resulting from accurate initiation at the adenovirus major late promoter. The dried gels were exposed for 45 min on Kodak XAR- 5 film with an intensifying screen. A, in addition to CTP (0.6 mM), UTP (0.6 mM), and [a-"P]GTP (0.012 mM, 32,000 cpm/pmol), the reaction contained ATP (0.6 mM) and/or AMP-PNP (0.6 mM) as indicated. dATP (0.02 mM) was added in reactions 2, 4,6, and 8. B, Reactions 1-6 contained CTP, UTP, [w3'P]GTP, and AMP-PNP with the addition of dATP (fane 2) , AMP (lane 3) , dCTP (lane 4) . dGTP (lane 5), or TTP (lane 6 ) a t 0.24 mM each. Reaction 7 contained CTP, UTP, GTP, AMP-PNP, and [ c ~ - ~ ' P ] ~ A T P (0.04 mM, 16,500 cpm/pmol).

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Specific Transcription Initiation Energy Requirement 5323

reaction is carried out in the presence of ATP, this ATP serves two different roles. First, as an RNA polymerase sub- strate, it is incorporated into the RNA, a role which can be filled by the imido derivative AMP-PNP. Second, ATP pro- vides an energy-rich phosphate bond, the hydrolysis of which is somehow required for specific transcription to take place. This energy requirement can alternatively be fulfilled by dATP, which indicates that the presumptive ATPase involved has little specificity for the sugar residue of the nucleotide molecule. In contrast, this ATPase shows a very strong pref- erence for an adenine ring, although the weak residual activity observed in the absence of ATP and dATP might indicate that one or more of the other three nucleotides present in the transcription reaction serves as a very weak substrate(s) for the ATPase (unless phosphate bond hydrolysis were only stimulatory, and not mandatory, for the specific transcrip- tion).

A T P Requirement for the Synthesis of Very Short RNA Transcripts-The generation of a run-off transcript from a specific promoter is a complex reaction that requires accurate initiation of RNA synthesis and efficient elongation of the transcript. A more detailed study of the ATP requirement for this reaction therefore necessitates the uncoupling of these steps, although there is presently no easy assay for transcrip- tion initiation (initiation being defined as the formation of the first phosphodiester bond). In order to discriminate be- tween initiation or promoter-proximal elongation events and subsequent elongation steps, we decided to take advantage of the fact that the first guanosine residue in the Ad2 ML transcript is the eleventh nucleotide (see Fig. 2 0 ) . The ab- sence of GTP should result in the synthesis of a decanucleo- tide, a reaction which should be independent of the overall elongation capability of the system. This approach could furthermore enable an independent study of the RNA chain elongation that should resume upon the delayed addition of the GTP substrate (if the transcription complexes, blocked in its absence, do not dissociate too rapidly).

Fig. 2 shows the characterization of the RNA transcripts generated from the Ad2 ML template in the absence of GTP in the reconstituted in vitro system. The direct analysis of these transcripts by electrophoresis on a sequencing gel (Fig. 2A) revealed an RNA with a mobility similar to that of an 8- or 9-nucleotide long oligomer (Band I ) ; this could be the expected decanucleotide since the presence of 5’ triphosphate would increase its electrophoretic mobility. This analysis also revealed an additional RNA migrating at the position of a 14- or 15-mer (Band II) , in addition to some minor higher molec- ular weight products. The presence of these unexpected RNAs could be explained by traces of contaminating GTP in the transcription reaction, which would allow some of the tran- scripts to be elongated past the first guanine residue. In accordance with this hypothesis, we observed that these dif- ferent RNAs accumulated with different kinetics, the larger ones appearing first and the putative decanucleotide appear- ing (for the most part) in a later phase, as if the contaminating GTP was then entirely depleted (results not shown). More- over, the final ratio of these different RNAs varied when different batches of the labeled nucleotide were used, some batches yielding near exclusive synthesis of t.he lower molec- ular weight transcript (see, for example, in Fig. 3); these results suggest that the [w”P]CTP or [(u-’~’P]UTP prepara- tions were the source of the contaminating GTP.

To further ascertain the nature of these transcripts, their size and GMP content were analyzed by treatment, respec- tively, with alkaline phosphatase and RNase T,. As illustrated in Fig. 2B, the smaller transcript (band I ) was resistant to

RNase T, (lune 3) and migrated as a decanucleotide following dephosphorylation (lane 2 ) . Dephosphorylation of the larger transcript (Bund 11) resulted in an oligonucleotide with the mobility expected for a quindecanucleotide (lune 51, while cleavage by RNase TI generated an RNA migrating at the expected position for an undecanucleotide with a 5”triphos- phate end (lune 6). Details of the sequence of these two RNAs were obtained by identification and quantitation of their degradation products after dephosphorylation and digestion by a mixture of RNases A and TI (14) (Fig. 2C). This analysis (see figure legend) demonstrated that the decanucleotide con- tained the sequences ACU, UU, and CC, each represented once; the sequence CU represented twice; and the sequence UC present three times; this is in perfect agreement with the first 10-nucleotide sequence of the ML transcript. The quin- decanucleotide gave the exact same degradation products, with two additional spots indicating the presence of the se- quences GC and AUC; these sequences are found in the ML RNA between the 11th and 15th residues. These results demonstrate that these two RNAs are transcripts initiated a t the ML cap site and terminated, respectively, before the first and second GMP residues.

All this implies that specific initiation at the ML cap site takes place accurately in the absence of GTP, the transcripts being elongated until the polymerase encounters the first cytosine residue on the DNA coding strand. As mentioned above, the 32P-labeled nucleotide that we used to label these short transcripts was sometimes contaminated by traces of GTP, so that some of the transcripts were elongated up to the second guanosine residue or even farther. In the absence of these labeled nucleotides, however, the transcripts produced should be a completely homogenous population of decanu- cleotides corresponding to the first 10 bases of the ML se- quence.

As shown in Fig. 3, the generation of these very short transcripts was subject to the same requirements as the 536- nucleotide long Sma I run-off transcript: their synthesis in the presence of ATP was inhibited either by heparin (lane I) or by a low concentration of @-amanitin (lane 2), and their synthesis was totally inhibited when the ATP was replaced by AMP-PNP (lane 7). Significantly, the addition of dATP (lune 8 ) restored transcription in the presence of AMP-PNP (as was the case for the synthesis of longer transcripts), indicating a requirement for hydrolyzable P-y phosphate bonds of an adenosine nucleotide. All of the previously iden- tified transcription factors were found necessary for this partial reaction, the efficiency of which was otherwise iden- tical with linear or closed circular DNA templates (not shown).

A remaining question was whether the 10-nucleotide long transcript that accumulated in the absence of GTP remained attached to the DNA template via the RNA polymerase or whether it was released with time. If the presumptive ternary transcription complex were stable, elongation should resume upon addition of the missing substrate. As shown in Fig. 3 (lane 5 ) , an additional 5 min of incubation in the presence of all four nucleotides at high concentration allowed all of the specific small RNAs that had been synthesized during 2 h of incubation in the absence of GTP to be chased into longer transcripts (upper arrow). The same observation was made when the first incubation had contained AMP-PNP and dATP in place of ATP (lane 9). This indicates that ternary transcription complexes with nascent RNA chains (10 nu- cleotides long) are stable under the conditions used for at least 2 h at 30 “C, and that even if the elongation is blocked

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

5324 Specific Transcription Initiation Energy Requirement

A

B I

16- I4 - 12- I I -

8 - 7- 6 - 5 -

C

-16 B a n d JI---

-12 - 1 1

B a n d I-. -- - 8 * -7 8 -6 " 5

I

[a"~] c T P

B a n d 1

D I

CP . UP

A-CP

[ d * P ] U T P

B a n d 1

I 1 I6

A-UP

[a!*P ] C T P

d a n d It

A C T C T C T T C C - G C A T C ' G . .

FIG. 2. Characterization of the small RNA transcripts generated from adenovirus major late pro- moter in the absence of the nucleotide GTP. A, transcription conditions were as in Fig. 1, except that the reaction contained only ATP (0.6 mM), UTP (0.025 mM), and [cY-~'P]CTP (0.025 mM, 32,000 cpm/pmol). After 2 h of incubation at 30 "C, the RNA was extracted with phenol-chloroform and analyzed, along with RNA molecular weight markers, on a 20% acrylamide 7 M urea sequencing gel. The arrows indicate the two major RNA transcripts with respective mobilities similar to those of 9- (Band I ) and 14-15- (Band 11) nucleotide long RNA fragments. The [32P]RNA markers were prepared by T, RNase digestion of Sma I run-off transcripts from adenovirus major late promoter (labeled at the cytosine residues) as described under "Materials and Methods." B, the oligonucleotides I and 11, synthesized as described above, were extracted from a preparative gel and analyzed by electrophoresis on a second sequencing gel both for their size after removal of their 5"triphosphate end by alkaline phosphatase digestion and for their GMP content by TI RNase cleavage. Lanes 1 and 4, Bands I and 11, respectively, untreated. Lanes 2 and 5, oligonucleotides I and 11, respectively, after alkaline phosphatase digestion. Lanes 3 and 6, Bands I and I1 after TI RNase digestion. C, The decanucleotide I, labeled with [c~-~'P]CTP (left) or [a-"PIUTP (middle) and the [a-3ZP]CTP-labeled pentadecanucleotide I1 (right) were extracted from a preparative gel, treated with alkaline phosphatase to remove the 5'-triphosphate, then digested to completion with a mixture of RNase A and T, and analyzed by two-dimensional chromatography on polyethylenimine miniplates (as described in Ref. 14). After autoradiography, the various spots were excised from the plates and the amount of radioactivity contained in each was quantified by liquid scintillation counting. The results indicated the following ratios of digestion products: for the [n-"PICTP-labeled decanucleotide, Up, Cp, ApCp (2.91.01.0); for the [a-"PjUTP-labeled decanucleotide Up, Cp, ApCp (0.9:2.4:1.0); and for the [a-32P]CTP-labeled pentadecanucleotide Up, Cp, ApCp, Gp, ApUp (3.2:1.01.1:0.9:0.8). D, nucleotide sequence of the Ad2 ML cap site.

by the absence of one of the substrates, the RNA transcript is not released.

Lack of ATP Requirement for RNA Chain Elongation- Since a homogenous population of stable ternary transcrip- tion complexes with very short RNA chains accumulates when t h e Ad2 ML template is transcribed in the absence of GTP, this system provides a convenient model for studying RNA chain elongation. In order to analyze the energy requirement for this elongation step, we first used chromatography on a gel filtration column (see the legend to Fig. 4) to remove the unincorporated nucleotides, and the ATP in particular, from such a transcription reaction. Heparin (20 pg/ml) was then

added to prevent new initiation events. As shown in Fig. 4, even with heparin present at a concentration that would be totally inhibitory if added before the beginning of transcrip- tion (see, for example, lane 1 of' Fig. 3), a further incubation of the excluded column fractions with the four polymerase substrates resulted in the synthesis of full length run-off transcripts. This indicates that these fractions contained transcription complexes, associated with the truncated DNA template, that resumed RNA chain elongation upon addition of the nucleotides. These fractions were totally devoid of endogenous ATP as ascertained by the absence of any elon- gation when the other three nucleotides only were added (lane

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Specific Transcription Initiation Energy Requirement 5325

A T P AMP-PNP 1 2 3 4

HEF? aA CHASE aA CHASE I 1 1 I I

dATP - - - + - + - + + I 2 3 4 5 6 7 8 9

: ' 5 4.

16-

12- I 1 - 8- 7-

- FIG. 3. The synthesis of very short RNA transcripts re-

quires hydrolysis of ATP or dATP. Transcriptions were carried out under standard conditions for 2 h a t 30 "C. The reactions (50 p l ) contained UTP (0.025 mM), [a-"PICTP (0.025 mM, 11,000 cpm/ pmol), and either ATP (0.6 mM, lanes 1-51 or AMP-PNP (1.2 mM, lanes 6-9). dATP (0.02 mM) was added in reactions 4, 6, 8, and 9. Lanes 2 and 6 are controls in which n-amanitin (nA) was present from 0 min, while reaction 1 contained heparin (20 pg/ml) added before the beginning of the incubation. In reactions 5 and 9, heparin (HEP.) (20 pg/ml) and all four nucleoside triphosphates (0.6 mM each) were added after 2 h of incubation, and the incorporated label was chased into longer RNA transcripts by a further 5 min of incubation at 30 "C. The transcripts were then phenol-extracted and analyzed on a 20% acrylamide-7 M urea sequencing gel. Autoradiog- raphy was for 54 h on Kodak BB-5 film with an intensifying screen.

I ) . However, in contrast to the results obtained for the com- plete transcription reaction, this run-off transcript synthesis from preinitiated transcription complexes took place regard- less of whether ATP (lane 2) or AMP-PNP (lane 3) was present as the adenosine nucleotide. The addition of dATP in the reaction with AMP-PNP had no effect (lane 4) . Thus, elongation is not implicated as an energy-dependent step in specific transcription by RNA polymerase 11, and the partic- ular transcription factor that requires ATP or dATP hydrol- ysis for its function must act at an earlier step of the reaction.

Interestingly, although the final amount of transcript was the same when the elongation was performed in the presence of AMP-PNP instead of ATP (as illustrated in the above experiment), the elongation rate, measured by monitoring the time course of appearance of the full length run-off transcript, was decreased 2-3-fold (probably due to the lower affinity of the polymerase for the analog as compared to the natural substrate) (data not shown). However, this decreased elon- gation rate did not affect the accumulation of run-off tran- scripts when initiation and elongation were linked, as in the experiment of Fig. 1. This implies that the elongation step is not rate limiting in the specific transcription reaction in uitro, a t least for the synthesis of 536-nucleotide adenovirus ML RNA.

Preliminary results have indicated that the rate-limiting

'soc ... FIG. 4. Lack of energy requirement for RNA chain elonga-

tion. A 50-pl transcription reaction containing ATP, CTP, and UTP (0.6 mM each) was incubated for 1 h a t 30 "C. The transcription complexes were then separated from the nucleotide substrates by gel filtration a t room temperature on a 1-ml agarose 0.5 m column equilibrated with transcription buffer. The excluded fractions were pooled and further incubated for 20 min a t 30 "C after addition of heparin (20 pglml), CTP (0.6 mM), UTP (0.6 mM), [a-"P]GTP (0.012 mM, 57,000 cpm/pmol), and either 0.6 mM of ATP ( l a n e 2) or 0.6 mM of AMP-PNP with ( l a n e 4 ) or without (lane 3) 0.04 mM of dATP. After phenol extraction, the transcripts were analyzed on a 4% acrylamide-Tris-borate-urea gel. The arrow indicates the position of the 536-nucleotide full length run-off transcript.

step of the in uitro reaction actually precedes initiation, and is ATP independent: thus, transcription started almost im- mediately after addition of the nucleotides when the tran- scription factors and the DNA template had been preincu- bated together in the absence of nucleotides for a sufficient period of time, and the presence of ATP (or dATP) during this preincubation did not influence the rate or extent of preinitiation complex formation (data not shown). On the other hand, we have been unable to uncouple the ATP- dependent step from the transcription initiation, which sug- gests that the energy-dependent step either occurs concomi- tantly with formation of the first few phosphodiester bonds, or is reversible in the absence of initiation.

Although we do not know the precise role of ATP hydrolysis in the early transcription event by RNA polymerase 11, there are numerous examples of ATP hydrolysis requirements for specific replication or transcription reactions. Thus, in pro- karyotes, several proteins involved in Escherichia coli or phage DNA replication were found associated with ATPase activi- ties (15) and the formation of an initiation complex between E. coli DNA polymerase 111 and a primed template is itself an ATP-dependent step (16). In eukaryotes, initiation of RNA synthesis in uitro by vaccinia virus cores has also been found to require ATP hydrolysis (17). We are now pursuing the purification of the transcription factor that seems implicated in the ATP-dependent step of specific transcription initiation by the human RNA polymerase I1 system. This should allow a more detailed analysis of the mechanism(s) involved.

REFERENCES

1. Shenk, T. (1981) Curr. Top. Microbiol. Immunol. 93, 25-46 2. Heinz, N., and Roeder, R. G. (1982) in Genetic Engineering

(Setlow, J. K., and Hollaender, A., eds), Vol. 4, pp. 57-82, Plenum Press, New York

3. Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979) Cell

4. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. 18,469-484

(1980) Proc. Natl. Acad. Sci. U. S. A. 77,3855-3859

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

5326 Specific Transcription Initiation Energy Requirement

5. Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J.

6. Slattery, E., Dignam, J. D., Matsui, T., and Roeder, R. G. (1983)

7. Samuels, M., Fire, A,, and Sharp, P. (1982) J. Biol. Chem. 257,

8. Dynan, W. S., and Tjian, R. (1983) Cell 32, 669-680 9. Davison, B. L., Egly, J. M., Mulvihill, E. R., and Chambon, P.

10. Bunick, D., Zandomeni, R., Ackerman, S., and Weinmann, R.

Biol. Chem. 255, 11992-11996

J . Biol. Chem. 258,5955-5959

14419-14427

(1982) Nature (Lond.) 301,'680-686

(1982) Cell 29,877-886

11.

12.

13. 14. 15.

16.

17.

Dignam, J. D., Lebowitz, R. M., and Roeder, R. G. (1983) Nucleic

Dignam, J. D., Martin, P., Shastry, B. S., and Roeder, R. G.

Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Volckaert, G., and Fiers, W. (1977) Anal. Biochem. 83, 228-239 Kornberg, A. (1976) in D N A Replication, pp. 277-317, W. H.

Burgers, P. M. J., and Kornberg, A. (1982) J. Biol. Chem. 257,

Gershowitz, A,, Boone, R. F., and Moss, B. (1978) J . Virol. 27,

Acids Res. 11, 1475-1489

(1983) Methods Enzymol. 101, 582-598

Freeman, San Francisco

11468-11473

399-408

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from

M Sawadogo and R G Roederpolymerase II system.

Energy requirement for specific transcription initiation by the human RNA

1984, 259:5321-5326.J. Biol. Chem. 

  http://www.jbc.org/content/259/8/5321Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/259/8/5321.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on June 13, 2018http://w

ww

.jbc.org/D

ownloaded from