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• W2015295275 abstract "Recent evidence, obtained in a reconstituted RNA polymerase II transcription system, indicated that the promoter escape stage of transcription requires template DNA located downstream of the elongating polymerase. In the absence of downstream DNA, very early elongation complexes are unable to synthesize transcripts longer than ∼10–14 nucleotides. In contrast, once transcripts longer than ∼15 nucleotides have been synthesized, an extended region of downstream DNA is no longer required (Dvir, A., Tan, S., Conaway, J. W., and Conaway, R. C. (1997) J. Biol. Chem. 272, 28175–28178). In this work, we sought to define precisely when, during the synthesis of the first 10–15 phosphodiester bonds, downstream DNA is required. We report that, for complete promoter escape, downstream DNA extending to position 40/42 is required. The polymerase can be forced to arrest at several points prior to the completion of promoter escape by removing downstream DNA proximally to positions 40/42. The positions at which the polymerase arrests appear to be determined by the length of available downstream DNA, with arrest occurring at a relatively fixed position of ∼28 nucleotides to the distal end of the template. A similar requirement is observed for transcription initiation,i.e. the formation of the first phosphodiester bond of nascent transcripts. In addition, we show that the requirement for a downstream region is independent of downstream DNA sequence, suggesting that the requirement reflects a general mechanism. Taken together, our results indicate (i) that downstream DNA is required continuously through the synthesis of the first 14–15 phosphodiester bonds of nascent transcripts, and (ii) that a major conformational change in the transcription complex likely occurs only after the completion of promoter escape. Recent evidence, obtained in a reconstituted RNA polymerase II transcription system, indicated that the promoter escape stage of transcription requires template DNA located downstream of the elongating polymerase. In the absence of downstream DNA, very early elongation complexes are unable to synthesize transcripts longer than ∼10–14 nucleotides. In contrast, once transcripts longer than ∼15 nucleotides have been synthesized, an extended region of downstream DNA is no longer required (Dvir, A., Tan, S., Conaway, J. W., and Conaway, R. C. (1997) J. Biol. Chem. 272, 28175–28178). In this work, we sought to define precisely when, during the synthesis of the first 10–15 phosphodiester bonds, downstream DNA is required. We report that, for complete promoter escape, downstream DNA extending to position 40/42 is required. The polymerase can be forced to arrest at several points prior to the completion of promoter escape by removing downstream DNA proximally to positions 40/42. The positions at which the polymerase arrests appear to be determined by the length of available downstream DNA, with arrest occurring at a relatively fixed position of ∼28 nucleotides to the distal end of the template. A similar requirement is observed for transcription initiation,i.e. the formation of the first phosphodiester bond of nascent transcripts. In addition, we show that the requirement for a downstream region is independent of downstream DNA sequence, suggesting that the requirement reflects a general mechanism. Taken together, our results indicate (i) that downstream DNA is required continuously through the synthesis of the first 14–15 phosphodiester bonds of nascent transcripts, and (ii) that a major conformational change in the transcription complex likely occurs only after the completion of promoter escape. general factor for transcription initiation by RNA polymerase II adenovirus 2 major late nucleotide(s) adenosine 5′-O-(thio)triphosphate 3′-O-methylguanosine 5′-triphosphate TATA-box binding protein Eukaryotic messenger RNA synthesis is a complex biochemical process that depends on RNA polymerase II and a variety of general and gene-specific transcription factors. Much information about the function of the RNA polymerase II transcription complex has been obtained in reconstituted, in vitro transcription systems, in which the contributions of individual cofactors to rate-limiting steps can be specifically evaluated (1Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Crossref PubMed Scopus (345) Google Scholar, 2Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (852) Google Scholar). Stable binding of RNA polymerase II to the promoter requires minimally the presence of the general transcription factors TFIID1 (or TBP), TFIIB, and TFIIF. Before transcription can begin, the double-stranded structure of the DNA template surrounding the initiation site on the promoter needs to be melted into single-stranded DNA in a process that is referred to as open complex formation (3Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar). Open complex formation and transcription initiation (i.e. the formation of the first phosphodiester bond (Refs. 4Luse D.S. Jacob G.A. J. Biol. Chem. 1987; 262: 14990-14997Abstract Full Text PDF PubMed Google Scholar, 5Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 6Dvir A. Garrett K.P. Chalut C. Egly J.M. Conaway J.W. Conaway R.C. J. Biol. Chem. 1996; 271: 7245-7248Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar)) depend on the presence of two additional general transcription factors, TFIIE and TFIIH, and are catalyzed by an ATP(dATP)-dependent DNA helicase activity associated with TFIIH (6Dvir A. Garrett K.P. Chalut C. Egly J.M. Conaway J.W. Conaway R.C. J. Biol. Chem. 1996; 271: 7245-7248Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Holstege F.C. van der Vliet P.C. Timmers H.T. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar, 8Moreland R.J. Tirode F. Yan Q. Conaway J.W. Egly J.M. Conaway R.C. J. Biol. Chem. 1999; 274: 22127-22130Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Bradsher J. Coin F. Egly J.M. J. Biol. Chem. 2000; 275: 2532-2538Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Transcription initiation is followed by a short phase that is referred to as promoter escape. This phase primarily includes the formation of the first 10–15 phosphodiester bonds of nascent RNA transcripts and is characterized by functional instability of the RNA polymerase II transcription complex (10Dvir A. Biochim. Biophys. Acta. 2002; 1577: 218-223Google Scholar). In the absence of either TFIIH or an ATP(dATP) cofactor, early RNA polymerase II elongation intermediates are prone to premature arrest at ∼10 to ∼14 base pairs downstream of the transcriptional start site (11Dvir A. Conaway R.C. Conaway J.W. J. Biol. Chem. 1996; 271: 23352-23356Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Dvir A. C C.R. W C.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar). In contrast, further transcript elongation by very early RNA polymerase II elongation intermediates that have successfully synthesized transcripts 14 or 15 nucleotides long requires neither TFIIH nor an ATP(dATP) cofactor. Finally, completion of promoter escape depends on the presence of an extended region of downstream DNA; digestion of a duplex template containing the AdML promoter with a restriction enzyme that cuts the template 35/39 nucleotides downstream of the DNA template has no effect on initiation but results in arrest by RNA polymerase II at a position 10–15 nucleotides downstream of the transcriptional start site. However, once polymerase has successfully synthesized ∼14 nucleotide transcripts, digestion of the template with the same restriction enzyme has no effect on further elongation by RNA polymerase II (13Dvir A. Tan S. Conaway J.W. Conaway R.C. J. Biol. Chem. 1997; 272: 28175-28178Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Based on these characteristics, we operationally define early RNA polymerase II elongation complexes that have synthesized transcripts ∼15 nucleotides or longer as those that have successfully escaped the promoter. During the transcription cycle, promoter escape follows immediately after initiation. There are a number of notable similarities between these two very early stages of transcription; both share a requirement for an ATP(dATP) cofactor and depend on the presence of TFIIE and TFIIH (6Dvir A. Garrett K.P. Chalut C. Egly J.M. Conaway J.W. Conaway R.C. J. Biol. Chem. 1996; 271: 7245-7248Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 11Dvir A. Conaway R.C. Conaway J.W. J. Biol. Chem. 1996; 271: 23352-23356Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Dvir A. C C.R. W C.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar, 14Kumar K.P. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9767-9772Crossref PubMed Scopus (66) Google Scholar), and both are suppressed by mutations in the same DNA helicase subunit of TFIIH, encoded by theXeroderma pigmentosum complementation group B(XPB) gene (8Moreland R.J. Tirode F. Yan Q. Conaway J.W. Egly J.M. Conaway R.C. J. Biol. Chem. 1999; 274: 22127-22130Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Bradsher J. Coin F. Egly J.M. J. Biol. Chem. 2000; 275: 2532-2538Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 16Yan Q. Moreland R.J. Weliky Conaway J. Conaway R.C. J. Biol. Chem. 1999; 274: 35668-35675Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), arguing that promoter melting is required for both transcription initiation and promoter escape and is likely catalyzed by the same mechanism in both steps. Finally, initiation, like promoter escape, depends on the presence of an extended downstream DNA region, which has been shown to extend to somewhere between 23 and 35 nucleotides downstream of the transcriptional start site (13Dvir A. Tan S. Conaway J.W. Conaway R.C. J. Biol. Chem. 1997; 272: 28175-28178Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Although previous studies have provided strong support for the model that interactions between component(s) of the transcription complex and downstream DNA are important for both initiation and promoter escape, a variety of important questions regarding the structure and function of downstream DNA remain unanswered. These include the following. (i) What are the precise boundaries of the regions required to support transcription initiation and promoter escape? (ii) Does the requirement for downstream DNA in promoter escape depend on downstream DNA sequence? (iii) Is the completion of promoter escape accompanied by a major rearrangement of the transcription complex? The experiments presented here provide evidence that the downstream DNA requirements do not change significantly through the synthesis of the first 14–15 bonds of nascent transcripts, that downstream DNA operates in a sequence-independent manner, and that a major conformational change in the transcription complex likely happens at the completion of promoter escape. The primary DNA template used in this study is M13mp19-AdML, which contains original AdML promoter sequences from −50 to 10 (12). AdML promoter mutants were prepared by the uracil-containing DNA method for site-directed mutagenesis (17Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar), using the Bio-Rad Mutagen 2 system. Resulting clones were verified by sequencing. For transcription, a 444-base pair fragment was produced by PCR from M13mp19-AdML. The primers were 5′-GACGGCCAGTGAATTCGA and 5′-CCAGCGTGGACCGCTTGC. The resulting DNA fragment, which contains sequences that extend 77 bp upstream and 367 bp downstream of the transcriptional start site, was gel-purified by agarose gel electrophoresis prior to use in transcription reactions. Hybrid templates were prepared containing the core T7 promoter fused to AdML promoter sequences with downstreamHaeIIII restriction sites. The T7 promoter sequence was introduced to the AdML templates by performing PCR using M13mp19-AdML derivatives as templates. We used an upstream PCR primer consisting of the T7 promoter: 5′(−25)-ATGGTACCTAATACGACTCACTATAGGGAGAACTCTCTTCCTCTAGAGTCG. The underscored sequence is a 20-base sequence complimentary to the AdML template strand at promoter positions 1–20. At the 5′ end of the T7 promoter, a six-base “clamp” sequence has been added (18Jorgensen E.D. Durbin R.K. Risman S.S. McAllister W.T. J. Biol. Chem. 1991; 266: 645-651Abstract Full Text PDF PubMed Google Scholar). The downstream primer was (5′-CCAGCGTGGACCGCTTGC), identical to that used in the AdML template amplification reactions. The M13mp19-AdML templates used had HaeIII sites positioned at 34, 40, and 44 relative to the AdML start site. Because T7 begins transcription at −6 relative to the AdML insertion, after digestion of templates withHaeIII the resulting transcripts are 40, 46, and 50 nt long. T7-AdML template amplification was carried out in 100 μl with 5 units of Taq DNA polymerase, 20 mm Tris-HCl, pH 8.0, 1.5 mm MgCl2, 50 mm KCl, 0.2 mm each of dNTPs (dATP, dCTP, dTTP, dGTP), 50.0 pmol of each primer, and 830 fmol of M13mp19-AdML template DNA. The length of the PCR product is 398 bp. PCR products were gel-purified by agarose gel electrophoresis. RNA polymerase II (19Serizawa H. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7476-7480Crossref PubMed Scopus (109) Google Scholar) and TFIIH (20Conaway R.C. Reines D. Garrett K.P. Powell W. Conaway J.W. Methods Enzymol. 1996; 273: 194-207Crossref PubMed Google Scholar) were purified from rat liver nuclear extracts as described. Recombinant yeast TBP (21Schmidt M.C. Kao C.C. Pei R. Berk A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7785-7789Crossref PubMed Scopus (115) Google Scholar, 22Conaway J.W. Hanley J.P. Garrett K.P. Conaway R.C. J. Biol. Chem. 1991; 266: 7804-7811Abstract Full Text PDF PubMed Google Scholar) and TFIIB (23Tsuboi A. Conger K. Garrett K.P. Conaway R.C. Conaway J.W. Arai N. Nucleic Acids Res. 1992; 20: 3250Crossref PubMed Scopus (41) Google Scholar) were expressed in Escherichia coli and purified as described. Recombinant TFIIE was prepared as described (24Peterson M.G. Inostroza J. Maxon M.E. Flores O. Admon A. Reinberg D. Tjian R. Nature. 1991; 354: 369-373Crossref PubMed Scopus (144) Google Scholar), except that the 56-kDa subunit was expressed in E. coli BL21(DE3)-pLysS. Recombinant TFIIF was purified as described (25Tan S. Conaway R.C. Conaway J.W. BioTechniques. 1994; 16: 824-828PubMed Google Scholar) from E. coli JM109(DE3) coinfected with M13mpET-RAP30 and M13mpET-RAP74. Transcription experiments are performed in vitro using a reconstituted transcription system that includes RNA polymerase II, the five general transcription factors (TBP, TFIIB, TFIIE, TFIIF, and TFIIH), and gel-purified promoter-DNA. Promoter DNA and proteins are combined in a final volume of 30 μl to form preinitiation complexes during a 30 °C, 45-min incubation (11Dvir A. Conaway R.C. Conaway J.W. J. Biol. Chem. 1996; 271: 23352-23356Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The incubation buffer contains (final concentrations are given) 0.3 mm HEPES-NaOH, pH 7.9, 25 mmTris-HCl, pH 7.9, 25 mm KCl, 4 mmMgCl2, 0.2 mm EDTA, 1 mmdithiothreitol, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl alcohol, and 6% (v/v) glycerol. Each reaction included 20 ng of AdML DNA fragment, 50 ng of recombinant yeast TBP, 10 ng of recombinant TFIIB, 20 ng of recombinant TFIIF, 20 ng of recombinant TFIIE, 150 ng of highly purified TFIIH, and 0.01 units of RNA polymerase II. Digestion of template DNA by PstI or HaeIII endonuclease was included in various phases of the transcription experiment by adding 2 μl of a solution containing 0.1–0.5 units of the respective enzymes. Digestions were carried at 30 °C for the times indicated, mostly 20 min. Separate experiments were carried to verify that these conditions allowed for complete digestions. Transcription was initiated by a labeling mix containing [α-32P]CTP (3,000 Ci/mmol), a CpU dinucleotide primer, ATP or dATP cofactor, and other ribonucleoside triphosphates as described in the figure legends. The final volume of reaction mixes was 35 μl. Transcription was carried at 30 °C for the time indicated in the figure legends. Transcription was stopped by addition of 15 μl of stop solution containing 100 mm EDTA. A 55-μl loading dye containing 10.0 m urea, 0.025% bromphenol blue, and 0.025% xylene cyanole was added to each sample. Samples were then heated to 90 °C for 3 min, briefly centrifuged to remove insoluble particles, and separated on a 25% acrylamide, 3% bisacrylamide, 6.0m urea gel as described (6Dvir A. Garrett K.P. Chalut C. Egly J.M. Conaway J.W. Conaway R.C. J. Biol. Chem. 1996; 271: 7245-7248Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and visualized by autoradiography. For run-off transcription experiments, reactions were stopped by addition of 0.05 m EDTA, 0.1 m NaCl, and 0.5% SDS to the reaction mixture. Precipitation is accomplished by ethanol followed by a 70% ethanol wash. Dried pellets were resuspended in 27 μl of solution of 10 m urea, 0.025% bromphenol blue, and 0.025% xylene cyanol FF, heated at 90 °C for 5 min, then loaded on urea-containing acrylamide gels for electrophoretic separation of transcripts. T7-AdML templates obtained by PCR above were digested withHaeIII prior to transcription by combining T7-AdML PCR product, 1 unit of HaeIII, and 1× enzyme buffer (50 mm Tris-HCl, pH 8.0, 10 mm MgCl2, and 50 mm NaCl) in a 10-μl reaction incubated for 15 min at 37 °C. The RNA markers were synthesized in vitro as follows; reactions of 50 μl included 50 units of T7 RNA polymerase, 1× enzyme buffer (40 mm Tris-HCl, pH 7.9, 6 mmMgCl2, 2 mm spermidine, 10 mmdithiothreitol), 0.1 mg/ml bovine serum albumin, 100 μmATP, 100 μm UTP, 100 μm GTP, 5 μm cold CTP, 67 nm[α-32P]CTP, and pre-digested T7-AdML DNA template. Reactions were incubated for 20 min at 37 °C. Reactions were stopped by using the same solutions, and protocols as are used to stop RNA polymerase II transcription reactions by the run-off protocol. To map precisely the extent of downstream DNA required at specific stages of early transcription, we generated a series of AdML promoter-containing constructs that contain restriction sites for the endonuclease HaeIII at various distances downstream of the transcription initiation site. The locations of the newly inserted cleavage sites correspond to promoter positions 26–50 from the start site in the AdML promoter (Fig. 1). When treated with HaeIII, these DNA templates acquire new, shorter ends that differ by 2-nucleotide increments from each other. In addition, following cleavage by HaeIII, templates are left with blunt ends, and the 3′ end of the templates differs from the sequence of the original, parental plasmid by at most 2 nucleotides. In our studies of early transcription by RNA polymerase II, we utilized a transcription system reconstituted with recombinant TBP, TFIIB, TFIIE, and TFIIF and purified polymerase and TFIIH from rat liver. Promoter-specific initiation was assayed by measuring synthesis of abortive, dinucleotide-primed trinucleotide transcripts. As shown previously, transcription initiation by RNA polymerase II from the AdML promoter can be primed by a variety of dinucleotides. These dinucleotides must be complementary to template DNA surrounding the transcriptional start site (26Samuels M.A. Fire A. Sharp P.A. J. Biol. Chem. 1984; 259: 2517-2525Abstract Full Text PDF PubMed Google Scholar). We assayed the synthesis of the first phosphodiester bond of nascent transcripts by measuring synthesis of trinucleotide transcripts in reactions containing the initiating dinucleotide CpU and [α-32P]CTP. These nucleotides support synthesis by polymerase of radioactively labeled CpUpC transcripts initiated at a position 3 base pairs upstream of the normal AdML transcriptional start site. To measure promoter escape, we monitor the synthesis of short transcripts in reactions containing the initiating dinucleotide CpU, ATP, UTP, [α-32P]CTP, as well as the RNA chain-terminating nucleotide 3′-O-methylguanosine triphosphate (3′-O-MeGTP). The maximal transcript length under these conditions is 18 nucleotides, determined by the insertion of 3′-O-MeGTP at the first G downstream of the initiation site. Because early RNA polymerase II elongation intermediates that have synthesized transcripts of ∼15 nucleotides or longer are considered to have successfully escaped the promoter, formation of the 3′-O-MeGTP terminated transcripts is a useful assay for the completion of promoter escape (11Dvir A. Conaway R.C. Conaway J.W. J. Biol. Chem. 1996; 271: 23352-23356Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Dvir A. C C.R. W C.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar, 13Dvir A. Tan S. Conaway J.W. Conaway R.C. J. Biol. Chem. 1997; 272: 28175-28178Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Previous experiments (13Dvir A. Tan S. Conaway J.W. Conaway R.C. J. Biol. Chem. 1997; 272: 28175-28178Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) have shown that initiation is strongly inhibited by cleaving an AdML promoter-containing template (pDN-AdML) withPstI, which cuts at position 23/27 and to a lesser degree by cleaving with SphI or HindIII, which cut at positions 29/33 and 35/39, respectively. However, these results only roughly defined the downstream border of the DNA required for initiation. In addition, because HindIII leaves a 5′ overhang and PstI leaves a 3′ overhang, the results of these earlier experiments could have been affected by the difference in DNA ends left by the different restriction enzymes used. In the experiments presented here, we used the abortive initiation assay described above to compare the activities of the Ad+26 through Ad+32 templates, with or without prior cleavage by HaeIII. As shown in Fig. 2, the promoters on all templates were capable of supporting abortive initiation when not cleaved with HaeIII, although several (Ad+30 and Ad+34) appeared somewhat less efficient. Following HaeIII cleavage, however, very little initiation occurred on the Ad+26 and Ad+28 templates. An intermediate level of initiation was observed followingHaeIII cleavage of the Ad+30, Ad+32, and Ad+34 templates, whereas HaeIII cleavage had little or no effect on initiation from the Ad+36 and Ad+40 templates. Thus, DNA downstream of position 34 is largely dispensable for initiation from the AdML promoter, and the region of downstream DNA most critical for initiation extends to 28 base pairs downstream of the transcriptional start site. To determine the extent of downstream DNA required for efficient promoter escape, templates that can be cleaved with HaeIII at positions from 34 to 46 base pairs downstream of the site of transcription initiation were tested for their abilities to support promoter escape, with and without prior treatment withHaeIII. As shown in Fig. 3, both transcription initiation and promoter escape were supported on all templates not treated with HaeIII. Cleavage of the Ad+42, Ad+44, and Ad+46 templates with HaeIII prior to transcription reactions had very little or no effect on the efficiency of promoter escape, as measured by synthesis of 18 nucleotide, 3′-O-MeG-terminated transcripts. Thus, DNA downstream of 42 is dispensable for promoter escape. In contrast, the efficiency of promoter escape was substantially reduced when the Ad+34, Ad+36, Ad+38, and Ad+40 templates were treated with HaeIII prior to the reactions, indicating that DNA extending to 40 is needed for efficient promoter escape. Notably, the length of the longest major transcripts synthesized following HaeIII cleavage depended on the position of the HaeIII cleavage sites. Thus, the lengths of the longest major transcripts were 8, 11, and 13–14 nt following cleavage with HaeIII at 34, 36, and 38, respectively, whereas cleavage at 40 allowed synthesis of a reduced level of 18-nt, 3′-O-MeG-terminated transcript. In Fig. 3, HaeIII digestion is performed before transcription initiation. To examine the effect of downstream DNA on promoter escape alone, we utilized a two-step, pulse-chase transcription protocol. In the first stage of the reaction, RNA synthesis was carried out in the presence of limiting concentrations of radioactive nucleotides, resulting in the formation of “pre-escaped” transcription complexes containing 3–9-nucleotide-long RNA transcripts. This initiation stage was followed by a chase phase, in which a large excess of unlabeled nucleotides was added, allowing further extension of transcripts and completion of promoter escape. Reaction conditions can be selectively changed between the two stages, allowing for the assessment of specific template and cofactor requirements for promoter escape. In the experiment of Fig. 4, we included a 15-min incubation with HaeIII to allow cleavage of downstream DNA between the pulse and chase stages of the reactions. This experiment was performed using promoters with restriction sites at positions 36, 38, 40, 42, and 44. Four transcription reactions were performed with each of the mutant templates; transcription in two of the reactions included only the labeling step, whereas in the other two it also included the cold chase phase. In each pair, one reaction was incubated with the HaeIII endonuclease at the end of the labeling stage. The other reaction served as a “noHaeIII” control. Transcripts at the end of the labeling stage of the reaction were 3–9 nucleotides long. The 3-nucleotide-long product is abortive, and the remaining bands corresponded primarily to 5- and 7-nucleotide-long transcripts. Addition of HaeIII to the “pulse-only” reactions did not significantly affect the length or amount of transcripts detected. The other two lanes shown for each template are reactions in which a cold nucleotide chase phase was added following the HaeIII treatment. Chase reactions were performed in the presence of ATP. As in the first two lanes, one reaction included a HaeIII treatment and the other was a “no HaeIII” control. In all the templates in the control lanes, promoter escape was supported to a similar level, as judged by the level of 18-nucleotide-long, 3′-O-MeGTP-terminated transcripts. In reactions that included a HaeIII treatment, significant differences appeared between the various templates. On templates with theHaeIII restriction sites at positions 36 and 38, promoter escape was substantially suppressed. The 40 promoter showed an intermediate level of inhibition, and the 42 promoter showed only slight inhibition. Promoter escape on the template with aHaeIII site at 44 was not affected by the endonuclease. Therefore, in the two-step promoter escape experiment, DNA up to position 40/42 from the transcription initiation site appears critical for efficient promoter escape. This result is in full agreement with the result obtained in the single-step promoter escape experiment (Fig.3). The experiment presented in Fig. 4 provides additional information regarding transcription complexes that became arrested at promoter-proximal positions. The reduction in the level of promoter escape is accompanied by an increase in the level of promoter proximal arrest. This is evident by a new pattern of arrested RNA transcripts 11–15 nucleotides in length formed in reactions containing templates cut at positions 36 through 42. Small, but highly reproducible differences in the size distribution of arrested transcripts formed on the various templates can be recognized. Arrested transcripts were primarily 11 and 12 nucleotides in length on the 36 promoter, 12 and 13 nucleotides for the 38 template, and 13 and 14 nucleotides for the 40 template. On the 42 template, which is only slightly affected byHaeIII treatment, arrested transcripts were primarily 14 and 15 nucleotides in length. Because transcription is primed with CpU at position −3 relative to the in vivo initiation site, the observed RNA length less 3 nucleotides corresponds to the precise location of the RNA polymerase catalytic site at the time when its progress was stopped. The distance between the end of the downstream DNA and the size of paused transcripts, as seen in Fig. 4, is ∼28 ± 2 nucleotides for all templates where escape was suppressed by HaeIII treatment. These results suggest that, during the individual steps of the promoter escape stage, the precise position of the polymerase is in phase with the distal end of the required downstream DNA. During promoter escape, the downstream DNA region does not function as a template for transcription as it is at a distance of more than 20 nucleotides downstream from the catalytic site of the polymerase. Instead, it is likely that the downstream region forms critical contacts with component(s) of the transcription complex. These interactions might directly involve TFIIH, based on evidence from UV-induced protein-DNA cross-linking studies (27Kim T.K. Ebright R.H. Reinberg D. Science. 2000; 288: 1418-1422Crossref PubMed Scopus (215) Google Scholar, 28Douziech M. Coin F. Chipoulet J.M. Arai Y. Ohkuma Y. Egly J.M. Coulombe B. Mol. Cell. Biol. 2000; 20: 8168-8177Crossref PubMed Scopus (64) Google Scholar) and functional studies (29Spangler L. Wang X. Conaway J.W. Conaway R.C. Dvir A. Proc. Natl. Acad. Sci. U. S. A. 2" @default.
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• W2015295275 title "Promoter Escape by RNA Polymerase II" @default.
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• W2015295275 doi "https://doi.org/10.1074/jbc.m210848200" @default.
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