INTRODUCTION

Initiation of eukaryotic messenger RNA synthesis is a complex process catalyzed by multisubunit RNA polymerase II and governed by the concerted action of a diverse collection of transcription factors. Biochemical studies have resolved transcription by RNA polymerase II into multiple stages that include (i) assembly and ATP-dependent activation of the preinitiation complex, which evidence suggests involves unwinding of promoter DNA surrounding the transcriptional start site by the TFIIH DNA helicase, (ii) transcription initiation, and finally (iii) escape of polymerase from the promoter and formation of the stable elongation complex (1-3).

Although substantial information is currently available on the mechanisms underlying assembly and ATP-dependent activation of the preinitiation complex and transcription initiation, relatively little is known about how RNA polymerase II escapes the promoter to form the stable elongation complex. In a recent study carried out with a reconstituted basal transcription system composed of recombinant TBP, TFIIB, TFIIE, and TFIIF and highly purified RNA polymerase II and TFIIH from rat liver, we observed that, shortly after initiating transcription, RNA polymerase II becomes highly susceptible to arrest in a promoter-proximal region 9-13 base pairs downstream of the transcriptional start site (4). Furthermore, we found that arrest by RNA polymerase II in this region is suppressed by TFIIH in an ATP-dependent reaction that may be catalyzed by the TFIIH DNA helicase (5), since basal transcription in the reconstituted system is insensitive to the TFIIH kinase inhibitor H-8 (6, 7).

In this report, we present evidence that, in addition to TFIIH and an ATP cofactor, efficient promoter escape by RNA polymerase II requires formation of an "escape-competent" transcriptional intermediate. Formation of this intermediate exhibits a transient requirement for template DNA 40-50 base pairs downstream of the transcriptional start site. Here we present these findings, which shed new light on the mechanisms underlying promoter escape and formation of the stable RNA polymerase II elongation complex.

EXPERIMENTAL PROCEDURES

Unlabeled ultrapure ribonucleoside 5-triphosphates, dATP, and 3-O-MeGTP¹ were purchased from Pharmacia Biotech Inc. [-³²P]CTP (>400 Ci/mmol) was obtained from Amersham Corp. Dinucleotides CpU and CpA and polyvinyl alcohol (type II) were from Sigma. Bovine serum albumin (Pentex fraction V) was purchased from ICN Immunobiologicals. Recombinant ribonuclease inhibitor (RNasin 1) was obtained from Promega.

RNA polymerase II (8) and TFIIH (rat  (9), TSK SP 5-PW fraction (10)) were purified as described from rat liver nuclear extracts. Recombinant yeast TBP (AcA 44 fraction (11, 12)) and TFIIB (rat  (13)) were expressed in Escherichia coli and purified as described. Recombinant TFIIE was prepared as described (14), except that the 56-kDa subunit was expressed in E. coli strain BL21(DE3)-pLysS. Recombinant TFIIF was prepared as described (15) from E. coli strain JM109(DE3) co-infected with M13mpET-RAP30 and M13mpET-RAP74.

Preinitiation complexes were assembled at the AdML promoter at 28 °C by a 45-min preincubation of 105-µl reaction mixtures containing 20 mM Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH 7.9), 60 mM KCl, 4 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl alcohol, 7% (v/v) glycerol, 18 units of RNasin, ~30 ng of the EcoRI to NdeI fragment from pDN-AdML (16), ~150 ng of recombinant yeast TBP, ~30 ng of recombinant TFIIB, ~60 ng of recombinant TFIIF, ~60 ng of recombinant TFIIE, ~450 ng of TFIIH, and ~0.03 unit of RNA polymerase II. Transcription was initiated by addition of nucleotides as indicated in the figure legends. For analysis of short transcripts, 15 µl of each reaction mixture were added to 6 µl of 0.5 mg/ml proteinase K in 100 mM EDTA. Following incubation at room temperature for 15 min, 25 µl of 10 M urea containing 0.025% bromphenol blue and 0.025% xylene cyanol FF were added to each sample. The samples were vortexed for 10 s, heated at 70 °C for 5 min, and subjected to 25% acrylamide, 3% bisacrylamide, 7 M urea polyacrylamide gel electrophoresis as described (17). For analysis of runoff transcripts, 35 µl of each reaction mixture were added to 35 µl of 0.5 mg/ml proteinase K and 0.6 mg/ml yeast tRNA in 200 mM Tris-HCl (pH 7.6), 300 mM NaCl, 25 mM EDTA, and 2% (w/v) SDS. Following incubation at room temperature for 15 min, transcripts were extracted once with phenol/chloroform and ethanol-precipitated. Samples were resuspended in 10 M urea containing 0.025% bromphenol blue and 0.025% xylene cyanol FF and subjected to 6% acrylamide, 0.8% bisacrylamide, 7 M urea gel electrophoresis. Gels were imaged by autoradiography or on a Molecular Dynamics PhosphorImager.

RESULTS AND DISCUSSION

In experiments investigating the mechanism of formation of the RNA polymerase II elongation complex in a basal transcription system composed of recombinant TBP, TFIIB, TFIIE, and TFIIF and purified polymerase and TFIIH from rat liver, we discovered that promoter escape by RNA polymerase II has a transient requirement for template DNA 40-50 base pairs downstream of the transcriptional start site. To investigate the role of downstream template DNA in promoter escape, we took advantage of the plasmid pDN-AdML (16), which contains AdML core promoter sequences from 50 to +10 inserted between the KpnI and XbaI sites in the polylinker of pUC-18. The EcoRI to NdeI fragment of pDN-AdML was used as DNA template in transcription reactions. The NdeI site is located ~250 base pairs downstream of the AdML transcriptional start site. As illustrated in Fig. 1, pDN-AdML can also be cleaved by the restriction enzymes PstI, SphI, HindIII, and HaeIII, which cut the template strand at sites 22, 29, 39, and 48 nucleotides downstream of the AdML transcriptional start site. By digesting the EcoRI to NdeI fragment of pDN-AdML with these restriction enzymes before transcription initiation or after synthesis of short transcripts, we could assess the requirements for downstream template DNA during initiation, promoter escape, and subsequent elongation by RNA polymerase II.

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To determine how much downstream template DNA is required for transcription initiation, we used the dinucleotide-primed abortive initiation assay. As shown previously, RNA polymerase II will utilize dinucleotides to prime synthesis of promoter-specific transcripts (16, 18-20). Transcription initiation from the AdML promoter can be primed by a variety of dinucleotides within a small region centered around the transcriptional start site (20); for example, in the presence of [-³²P]CTP and the dinucleotide CpU, which supports initiation at a position 3 base pairs upstream of the AdML transcriptional start site, RNA polymerase II will initiate and synthesize the radioactively labeled trinucleotide CpU^*pC (see Fig. 1).

To determine how much downstream template DNA is required for very early elongation and promoter escape, we carried out transcription in the presence of CpU, ATP, UTP, [-³²P]CTP, and the chain-terminating nucleotide analog 3-O-MeGTP. Under these conditions, RNA polymerase II can initiate and synthesize transcripts that have a maximum length of 18 nucleotides (terminated at the first G residue, marked with an asterisk in Fig. 1).

In the experiment of Fig. 2, preinitiation complexes were assembled at the AdML promoter by preincubation of the EcoRI to NdeI fragment of pDN-AdML with RNA polymerase II and initiation factors. Following treatment of preinitiation complexes with either HaeIII, HindIII, SphI, or PstI, reaction mixtures were divided into three equal portions and assayed for synthesis of CpU-primed trinucleotide transcripts, for synthesis of CpU-primed 3-O-MeG-terminated 18-nucleotide-long transcripts, and, to control for efficient restriction enzyme digestion of the template, for synthesis of CpU-primed full-length runoff transcripts. As shown in lanes 15 and 19, transcription of the undigested KpnI to NdeI fragment of pDN-AdML resulted in synthesis of the full-length ~250-nucleotide-long runoff transcript. Synthesis of the NdeI-terminated ~250-nucleotide runoff transcript was almost completely abolished by treatment of preinitiation complexes with HaeIII, HindIII, SphI, or PstI (lanes 16-18, 20, and 21), arguing that the DNA templates were digested to near completion by each restriction enzyme.

3-OMeG

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Synthesis of CpU-primed trinucleotide transcripts was unaffected by digestion of preinitiation complexes with HaeIII (compare lanes 1 and 3) and only modestly reduced by digestion with either HindIII (compare lanes 1 and 5) or SphI (compare lanes 1 and 7 and lanes 9 and 10). In contrast, synthesis of trinucleotide transcripts was almost completely inhibited by digestion of preinitiation complexes with PstI (compare lanes 9 and 11). Thus, transcription initiation by RNA polymerase II does not require template DNA downstream of +29, but is strongly dependent on the presence of template DNA between +23 and +28, even though the preinitiation complex does not protect DNA in this region from digestion by restriction enzymes.

Like synthesis of CpU-primed trinucleotide transcripts, synthesis of CpU-primed 3-O-MeG-terminated 18-nucleotide-long transcripts was unaffected by digestion of preinitiation complexes with HaeIII prior to initiation (compare lanes 2 and 4). In contrast, little or no 3-O-MeG-terminated 18-nucleotide transcripts were synthesized when preinitiation complexes were digested with HindIII (lane 6) or SphI (lanes 8 and 13). Under these conditions, CpU-primed transcripts reached a maximum length of only ~10-12 nucleotides. Thus, promoter escape by very early RNA polymerase II elongation complexes is strongly dependent on the presence of template DNA located between 40 and 47 base pairs downstream of the AdML transcriptional start site (or between 30 and 40 base pairs downstream of the polymerase catalytic site), even though this region of the DNA template is not essential for assembly of the preinitiation complex formation and transcription initiation.

Although digestion of preinitiation complexes with HindIII prior to transcription initiation is sufficient to inhibit synthesis of transcripts longer than ~10-12 nucleotides, digestion of preinitiation complexes with HindIII after synthesis of ~14-nucleotide-long transcripts does not prevent their further extension. In the experiment of Fig. 3, preinitiation complexes were assembled at the AdML promoter by preincubation of the EcoRI to NdeI fragment of pDN-AdML with RNA polymerase II and initiation factors. Transcription was carried out in the presence of the initiating dinucleotide CpA, [-³²P]CTP, UTP, and dATP to satisfy the energy requirement for transcription initiation. Under these conditions, RNA polymerase II can initiate and synthesize transcripts that have a maximum length of 14 nucleotides (terminated at the U residue immediately preceding the first A residue that must be incorporated into the transcript, marked with a circle in Fig. 1). Following treatment of preinitiation complexes with either HaeIII or HindIII, RNA polymerase II transcription intermediates were assayed for their abilities to extend the CpA-primed 14-nucleotide-long transcripts to 3-O-MeG-terminated 16-nucleotide transcripts or to full-length runoff transcripts. As shown in lanes 1-6, digestion of preinitiation complexes with either HaeIII or HindIII had no effect on the efficiency with which CpA-primed 14-nucleotide-long transcripts were chased into 3-O-MeG-terminated 16-nucleotide transcripts. In addition, full-length runoff transcripts of the expected length were synthesized when nascent transcripts were chased into longer products in the presence of all four ribonucleoside triphosphates (lanes 7-9). Similar results were obtained when transcription was initiated with the dinucleotide CpU (data not shown).

3O-MeG

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As summarized in Fig. 4, in the process of investigating the mechanism of formation of the stable RNA polymerase II elongation complex, we have discovered that promoter escape by RNA polymerase II has a transient requirement for template DNA between 40 and 50 base pairs downstream of the transcriptional start site. We observe that, in the absence of downstream DNA in this region, very early RNA polymerase II elongation complexes are unable to synthesize transcripts longer than ~10-12 nucleotides. In contrast, elongating RNA polymerase II does not normally require an extended region of downstream template DNA for transcription; indeed, RNA polymerase II is able to transcribe to the extreme 3-end of most DNA templates during synthesis of longer runoff transcripts (21-23). Moreover, we observe that template DNA downstream of +40 is not required for assembly of the preinitiation complex, for initiation and synthesis of the first 10-12 phosphodiester bonds of nascent transcripts, or for further extension of transcripts longer than ~14 nucleotides. Taken together, our findings argue that promoter escape requires that the RNA polymerase II initiation complex undergoes a critical structural transition that is likely driven by interaction of one or more components of the transcriptional machinery with template DNA 40-50 base pairs downstream of the transcriptional start site and that results in formation of an escape-competent transcriptional intermediate.

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Finally, it is not yet clear which components of the RNA polymerase II transcriptional machinery require downstream DNA for promoter escape. In light of our recent findings (i) that RNA polymerase II is highly susceptible to arrest in a very similar promoter-proximal region (~9-13 base pairs downstream of the transcriptional start site) during promoter-specific transcription reconstituted with TBP, TFIIB, TFIIE, TFIIF, and TFIIH (4) and (ii) that arrest by polymerase at this site is suppressed by TFIIE and TFIIH in a step that requires an ATP cofactor (5), it is tempting to speculate TFIIE, TFIIH, ATP, and downstream DNA are all involved in formation of the same escape-competent transcriptional intermediate. In this regard, it is noteworthy that results of previous footprinting (10, 24-26) and electron crystallographic studies (27) suggest that TFIIH and/or TFIIE may be positioned at the leading edge of the preinitiation complex, consistent with the possibility that they could facilitate promoter escape through interactions with downstream DNA.