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Three transitions in the RNA polymerase II transcription complex during initiation

1997; Springer Nature; Volume: 16; Issue: 24 Linguagem: Inglês

10.1093/emboj/16.24.7468

1460-2075

Frank C. P. Holstege,

Genomics and Chromatin Dynamics

Article15 December 1997free access Three transitions in the RNA polymerase II transcription complex during initiation Frank C. P. Holstege Frank C. P. Holstege Search for more papers by this author Ulrike Fiedler Ulrike Fiedler Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA, Utrecht, The Netherlands Search for more papers by this author H. Th. Marc Timmers Corresponding Author H. Th. Marc Timmers Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA, Utrecht, The Netherlands Search for more papers by this author Frank C. P. Holstege Frank C. P. Holstege Search for more papers by this author Ulrike Fiedler Ulrike Fiedler Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA, Utrecht, The Netherlands Search for more papers by this author H. Th. Marc Timmers Corresponding Author H. Th. Marc Timmers Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA, Utrecht, The Netherlands Search for more papers by this author Author Information Frank C. P. Holstege2, Ulrike Fiedler1 and H. Th. Marc Timmers 1 1Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA, Utrecht, The Netherlands 2Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA, 02142 USA *E-mail: [email protected] The EMBO Journal (1997)16:7468-7480https://doi.org/10.1093/emboj/16.24.7468 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have analyzed transcription initiation by RNA polymerase II (pol II) in a highly efficient in vitro transcription system composed of essentially homogeneous protein preparations. The pol II complex was stalled on adenovirus major late promoter templates at defined positions, and the open region and RNA products of these complexes were examined. The first transition is formation of the open complex, which can be reversed by addition of ATPγS. The open region is no longer sensitive to ATPγS after formation of a four-nucleotide RNA, which constitutes the second transition. This indicates that the ATP-dependent DNA helicase activity of TFIIH is required to maintain the open region only during formation of the first three phosphodiester bonds. The downstream part of the transcription bubble expands in a continuous motion, but the initially opened region (−9/−2 on the non-template strand) recloses abruptly when transcription reaches register 11. This third transition is accompanied by a switch from abortive to productive RNA synthesis, which implies promoter clearance. Our findings provide a framework to analyze regulation of these specific transitions during transcription initiation by pol II. Introduction Regulating the rate of transcription initiation by RNA polymerase is a major mechanism by which cells establish the proper expression patterns of their genes. Many proteins in eukaryotic cells are concerned with controlling the access of RNA polymerase II (pol II) to promoter regions of protein-encoding genes (for reviews, see Pirotta, 1995; Pazin and Kadonaga, 1997a,b). The establishment of in vitro transcription reactions and biochemical fractionations has led to the identification of the set of five basal transcription factors, which are minimally required to support accurate transcription initiation by pol II (for reviews, see Orphanides et al., 1996; Roeder, 1996). Recent evidence indicates that pol II and the basal transcription factors are not floating around freely in the nucleus, but that they are pre-assembled into pol II holoenzyme complex(es) together with many other proteins (for reviews, see Koleske and Young, 1995; Halle and Meisterernst, 1996). The composition of these large complexes (>2000 kDa) differs depending on the isolation procedure (Kim et al., 1994; Koleske and Young, 1994; Ossipow et al., 1995; Chao et al., 1996; Maldonado et al., 1996), and this reflects a tendency to dissociate during purification. Analyses of the different holoenzyme preparations show in all cases tested that the complete set of five basal transcription factors (TBP, TFIIB, TFIIE, TFIIF and TFIIH) need to be present to support in vitro transcription by pol II (Kim et al., 1994; Koleske and Young, 1994; Chao et al., 1996; Maldonado et al., 1996). This implies that the basal transcription factors perform unique roles in transcription initiation by pol II. Biochemical analyses have established the pathway of formation of the pol II pre-initiation complex from its isolated components (for reviews, see Orphanides et al., 1996; Roeder, 1996). In order to recognize promoter DNA, pol II requires the assistance of TBP (TATA-binding protein), TFIIB and TFIIF. In cellular extracts, TBP is found in complexes with other proteins. These TBP-associated factors (TAFs) play a role in transcriptional activation pathways and polymerase specificity, but for the basal pol II transcription reaction TBP alone suffices (for reviews, see Burley and Roeder, 1996; Verrijzer and Tjian, 1996). The TBP–TATA complex allows association of TFIIB, and together this forms the platform for promoter recognition by the TFIIF–pol II complex. Topological stress (Parvin and Sharp, 1993; Tyree et al., 1993; Goodrich and Tjian, 1994; Holstege et al., 1995) or a pre-melted region in promoter DNA (Pan and Greenblatt, 1994; Tantin and Carey, 1994; Holstege et al., 1996) are required to observe transcription initiation with this so-called DBpolF complex, indicating that it has a very limited capacity to melt the DNA strands to allow initiation. The basal transcription factors TFIIE and TFIIH are not essential for promoter recognition but are involved in promoter opening. TFIIE can stimulate transcription from supercoiled templates in the absence of TFIIH, and this is linked to the helical stability of promoter DNA (Holstege et al., 1995). On relaxed DNA templates, TFIIE is essential for TFIIH recruitment to the pre-initiation complex (Flores et al., 1992). Egly and co-workers found that two of the nine TFIIH subunits are ATP-dependent DNA helicases (Schaeffer et al., 1993, 1994). It was shown in activated transcription assays by Gralla and co-workers that ATP hydrolysis is linked directly to promoter melting as determined by potassium permanganate sensitivity assays, which map single-stranded regions in DNA (Wang et al., 1992). Using this assay and a highly purified basal transcription system, we obtained direct evidence that promoter opening requires the action of the ATP-dependent DNA helicase(s) of TFIIH (Holstege et al., 1996). With the adenovirus major late promoter (AdMLP), it was shown that a region predominantly upstream of the start site (−9/+2) becomes single stranded (Holstege et al., 1996; F.C.P.Holstege and H.T.M.Timmers, in preparation). The formation of the −9/+2 open complex occurs prior to formation of the first phosphodiester bond, and the ATP dependence of this reaction agrees well with earlier studies showing that ATP hydrolysis is required at an early phase of transcription initiation (Sawadogo and Roeder, 1984; Luse and Jacob, 1987; Luse et al., 1987; Conaway and Conaway, 1988). Further expansion of the transcription 'bubble' requires transcription initiation (Wang et al., 1992; Holstege et al., 1996). The molecular events between open complex formation and promoter clearance by pol II have not been determined in precise detail. The strong protein–protein interactions which are responsible for assembly of the pre-initiation complex need to be broken during the promoter clearance step. This step also marks the transition to the productive elongation mode of the transcription complex. In an elegant set of experiments, Reinberg and co-workers determined the positions of basal factor release from the initiation complex (Zawel et al., 1995). The role of ATP hydrolysis in promoter clearance is a controversial issue. Besides its ATP-dependent DNA helicase activity, TFIIH harbors a protein kinase specific for the C-terminal domain (CTD) of the largest subunit of pol II (for review, see Svejstrup et al., 1996). Stimulation of promoter clearance by the CTD kinase of TFIIH may be promoter specific (Serizawa et al., 1993; Akoulitchev et al., 1995; Mäkelä et al., 1995; Jiang et al., 1996) and depends on the composition of the transcription system (Serizawa et al., 1993; Li and Kornberg, 1994). Other studies have implicated the DNA helicase activity of TFIIH in promoter clearance (Goodrich and Tjian, 1994; Dvir et al., 1996b). In this study, we have used a highly purified and highly efficient in vitro system to examine the early phase of transcription initiation by pol II. DNA templates under control of the AdMLP were modified to stall pol II at defined positions. We have coupled the potassium permanganate sensitivity analysis of open regions in the DNA template to a direct analysis of the synthesized RNA products. This coupling allowed a careful description and provides a detailed model for the transcription initiation reaction by pol II. Results Our previous analyses of open complex formation indicate that prior to transcription initiation, the DNA strands of the AdMLP become single stranded from position −9 to +1 by the action of the ATP-dependent DNA helicase activity of TFIIH (Holstege et al., 1996). Additional experiments showed that position +2 is also susceptible to permanganate (F.C.P.Holstege and H.T.M.Timmers, in preparation) and, therefore, the initially opened region of the AdMLP encompasses positions −9 to +2. Concomitant with formation of the first phosphodiester bond, we observed a large expansion of the open region to position +8, and we named this expansion the second step in promoter opening (Holstege et al., 1996). When we analyzed opening of other pol II promoters (HIV, AdE4 and MMTV), expansions beyond the site of RNA synthesis were not observed (F.C.P.Holstege and H.T.M.Timmers, in preparation). In addition, the large expansion observed with the AdMLP was formed slowly and was increased by relatively high concentrations of CTP (>20 μM; F.C.P.Holstege and H.T.M.Timmers, unpublished data). Read-through has been observed during initiation of transcription by bacterial RNA polymerase (Carpousis and Gralla, 1980). Although other explanations are possible, we wondered whether the large expansion observed with the AdMLP is caused by pol II molecules transcribing beyond register 2 [we refer to register as the site of addition of the last nucleoside monophosphate (NMP) to the nascent RNA chain and we refer to position as the base pair of the DNA template relative to the transcription start site]. Bacterial RNA polymerase in the abortive transcription mode has a high rate of misincorporation (Metzger et al., 1993). To investigate this hypothesis, we used the RNA chain terminator 3′-O-methyl-GTP (3′-OMeGTP) to block read-through. In addition, to stall pol II in specific registers, we constructed a series of AdMLP templates with G residues at varying positions (Figure 1). Figure 1.DNA sequence of the non-template strand of the pDNAdML+nG plasmids. The sequence of the transcription start and stall site of the different AdMLP templates used in this study is depicted. Indicated in bold are the natural transcription start site (A) and the first G residue in the RNA chain. The templates are identical outside the region shown. Download figure Download PowerPoint Mutant AdMLP templates were labeled on the non-template strand and used in potassium permanganate sensitivity assays as described earlier (Holstege et al., 1996; Holstege and Timmers, 1997). Briefly, initiation complexes were formed on AdMLP fragments by pre-incubation with homogenous preparations of TBP, TFIIB, TFIIE and TFIIF, which were expressed in bacteria or in insect cells with recombinant baculoviruses (TFIIF) (see also Holstege et al., 1996). The pol II preparation was obtained from calf thymus extracts by a combination of ion-exchange and immunoaffinity chromatography. The TFIIH preparation was isolated from HeLa cells by an optimized protocol involving six purification steps. All protein preparations were free of contamination by other basal factor or pol II activities, as shown by leave-out experiments, and were devoid of nucleases, topoisomerases or co-factors like PC4 as determined by a number of assays (see Materials and methods and Holstege et al., 1996). The amounts of DNA template, basal factors and pol II used in the permanganate assay were titrated carefully in gel shift experiments to reach saturation of the template fragment. After pre-initiation complex formation, transcription was initiated by the addition of different combinations of ribonucleotides. In these assays, the efficiency of template usage for productive transcription is 25% at 4 fmol of template (see below). The downstream part of the transcription bubble expands continuously whereas the upstream part recloses discontinuously Figure 2 shows the results of the permanganate analysis using different mutant AdMLP templates, with the position of the first G residue indicated above the lanes. In all cases, addition of ATP to the reaction leads to increased sensitivity to permanganate of positions −9, −8, −5 and −2 (Figure 2A, lanes 2, 5, 8, 11, 14 and 17). Inclusion of 3′-OMeGTP with the AdML+2G fragment, which allows formation of only one phosphodiester bond, does not result in a downstream expansion of the open region (Figure 2A, compare lanes 2 and 3) as would be expected from our earlier study (Holstege et al., 1996). When pol II proceeds to register 3, a small increase in the reactivity of the −2T is observed (Figure 2A, compare lanes 5 and 6). Stalling pol II in register 4 further increases the sensitivity of position −5 and −2, and new sensitivities at +3 and +5 are induced (Figure 2A, lane 9). No further expansions are observed with AdML+6G (Figure 2A, lane 12) but, as expected, with AdML+9G positions +7 and +8 become sensitive to permanganate (Figure 2A, lane 15). As a control, we included the AdMLP fragment with the first G residue at +15 in this experiment (Figure 2A, lane 16–19). In agreement with our earlier study (Holstege et al., 1996), addition of ATP and CTP results in the large expansion to position +8 (Figure 2A, lane 18). This is well beyond the expected stall site in register 3, which specifies addition of a UMP residue (see Figure 1), and strongly suggests that our previously reported expansion upon formation of the first phosphodiester bond is due to read-through by the pol II enzyme (see also below). Inclusion of ATP, CTP, UTP and 3′-OMeGTP in the AdML+15G reaction results in stalling of pol II at register 15 (Figure 2A, lane 19). This shows that the initially opened region from position −9 to −2 is no longer sensitive to permanganate and that the open region now encompasses position +3 to +13. These results show that in the pol II initiation complex, downstream melting of the template DNA keeps abreast with RNA synthesis. The downstream edge of the open region is within 1 bp from the register of RNA synthesis. Figure 2.The transcription bubble expands downstream continuously and recloses upstream discontinuously. (A) The results of permanganate sensitivity assays with pol II stalled at different positions. Transcription initiation complexes were assembled with AdML+2G (lanes 1–3), AdML+3G (lanes 4–6), AdML+4G (lanes 7–9), AdML+6G (lanes 10–12), AdML+9G (lanes 13–15) and AdML+15G (lanes 16–19) promoter fragments as described in Materials and methods. Reactions received either no nucleotides as a control (lanes 1, 4, 7, 10, 13 and 16), 60 μM ATP (lanes 2, 5, 8, 11, 14 and 17), 60 μM ATP and 120 μM 3′-OMeGTP (lane 3), 60 μM ATP, 10 μM CTP and 120 μM 3′-OMeGTP (lane 6), 60 μM ATP, 10 μM CTP, 10 μM UTP and 120 μM 3′-OMeGTP (lanes 9, 12, 15 and 19) or 60 μM ATP and 10 μM CTP (lane 18). After a 5 min incubation at 30°C, potassium permanganate was added for 90 s and the reaction was processed as described in Materials and methods. Positions of reactive thymidines are determined by co-migration of a G+A ladder of the AdML+11G fragment (data not shown) and are indicated to the right. The arrow marks the start site and direction of transcription. It should be noted that due to the T→G mutation no reactivity at the +3 position can be expected with the AdML+3G template in lane 6. (B) The initially melted open region (positions −9 to −2) recloses during translocation of pol II from register 9 to register 11. The open regions of transcription complexes stalled at the +9 (lane 2), +10 (lane 4), +11 (lane 6) and +15 (lane 8) positions were determined by permanganate sensitivity assays as described in (A). Reactions were reconstituted using AdML+9G (lanes 1 and 2), AdML+10G (lanes 3 and 4), AdML+11G (lanes 5 and 6) or AdML+15G (lanes 7 and 8) promoter fragments. Reactions received either no nucleotides (lanes 1, 3, 5 and 7) or 60 μM ATP, 10 μM CTP, 10 μM UTP and 120 μM 3′-OMeGTP (lanes 2, 4, 6 and 8). After a 5 min incubation at 30°C, potassium permanganate was added for 90 s and the reaction was processed as described in Materials and methods. The positions of reactive thymidines as determined with a co–migrating G+A ladder (data not shown) are indicated to the right. The arrow marks the start site and direction of transcription. It should be noted that due to the T→G mutation, no reactivity at the +11 position can be expected with the AdML+11G template in lane 6. Download figure Download PowerPoint In the analysis of Figure 2A, the initially opened region (−9/−2), which is melted by the action of the TFIIH DNA helicase(s), recloses when pol II transcribes from register 9 to 15. To map the reclosure more precisely, two additional AdMLP constructs were analyzed. Figure 2B (compare lanes 2 and 6) shows that positions −9, −8, −5 and −2 are not reactive to permanganate when pol II is stalled in register 11, and this is similar to the reaction with the AdML+15G template (Figure 2B, lane 8). The AdML+10G fragment represents an intermediate situation (Figure 2B, compare lanes 2, 4 and 6). These results indicate that between register 9 and 11, the pol II initiation complex undergoes a conformational change, which results in reclosure of the −9/−2 region. Thus, while the downstream part of the open region expands in a continuous motion, the upstream part of the open region readopts the double-stranded conformation discontinuously. Analysis of RNAs formed by stalled transcription complexes To investigate whether pol II is stalled in the expected register, we analyzed the RNA products formed under conditions of the permanganate sensitivity assay. Initiation complexes were assembled on different AdMLP templates as previously, and transcription was initiated by the addition of 60 μM ATP, 10 μM [α-32P]CTP, 10 μM UTP and 120 μM 3′-OMeGTP as indicated above the lanes. After 5 min of incubation, the reactions were stopped and analyzed. Figure 3A shows that in the absence of the RNA chain terminator, 3′-OMeGTP read-through products are observed with the AdML+4G, +6G, +9G, +10G, +11G and +15G templates (lanes 4–9). Longer exposure reveals the presence of read-through products in lanes 2 and 3 (data not shown). In several cases, the read-through products are one nucleoside longer than the expected product (4 nt in lane 4, 10 nt in lane 7, 11 nt in lane 8 and 15 nt in lane 9), which may indicate that these RNAs are elongated inefficiently due to mispairing of the last residue with the DNA template. The inclusion of 3′-OMeGTP in the reactions results in the production of a single RNA product in the cases of the AdML+10G, +11G and +15G templates (Figure 3A, lanes 14–16). While the expected RNA is the major species with the AdML+6G and +9G templates, products one nucleoside shorter are also observed (Figure 3A, lanes 12 and 13). With the AdML+3G and +4G templates, the expected product is formed with an efficiency similar to the one-nucleotide shorter product (Figure 3A, lanes 10 and 11). This suggests an inefficient incorporation of 3′-OMeGTP in these cases, which may lead to release of the nascent RNA from the pol II complex. For bacterial RNA polymerase, it is known that efficiency of NMP addition to a growing RNA chain is dependent on the specific NMP and on the sequence of the template (Levin and Chamberlin, 1987). Inclusion of 2 μg/ml α-amanitin in the reaction completely inhibits RNA synthesis, which verifies that RNAs are formed by pol II (data not shown). From the analysis of Figure 3A, we conclude that addition of 3′-OMeGTP to the reactions results in a more homogenous population of RNA products. Figure 3.Analysis of RNA products formed with mutant AdMLP templates. (A) Initiation complexes were assembled on mutant AdMLP templates (as indicated at the top of the figure) similarly to in the permanganate sensitivity assays. Nucleotide triphosphates were added, as indicated above the lanes, for 5 min. Subsequently, RNA products were processed and analyzed by denaturing polyacrylamide gel electrophoresis as described in Materials and methods. Reactions received either no promoter DNA fragment (lane 1), AdML+15G (lanes 2, 9 and 16), AdML+3G (lanes 3 and 10), AdML+4G (lanes 4 and 11), AdML+6G (lanes 5 and 12), AdML+9G (lanes 6 and 13), AdML+10G (lanes 7 and 14) or AdML+11G promoter fragment (lanes 8 and 15). All reactions were reconstituted with 20 fmol of DNA template and initiated with 60 μM ATP and 10 μM [α-32P]CTP. In addition, reactions also received 10 μM UTP (lanes 4–9), 120 μM 3′-OMeGTP (lane 10) or 10 μM UTP and 120 μM 3′-OMeGTP (lanes 1 and 11–16). The size of each product (in nucleosides) is indicated by the number above the band, except for the two-nucleoside product in lane 10 for which the number is indicated below the band. Products which contain 3′-O-MeGMP are indicated by the letter G. Therefore, the product indicated by the number '3′ in lane 4 is ApCpU, whereas the product in lane 10 indicated by '3G' is ApCpG-3′-O-methyl. All reactions were treated with alkaline phosphatase to remove phosphate groups at the 5′-end of the RNAs. Besides reducing the background reactivity in each lane, this significantly decreases the migration rate of the smaller (five nucleosides or less) products. For these products, the migration rate initially increases with size. Substitution of a pyrimidine with guanosine results in a significant decrease in the migration rate (compare '3′ with '3G' in lanes 4 and 10). From RNA products longer than five nucleosides, the relative migration rate is as expected. The positions of the smaller products 2, 3G and 4G were verified by UV shadowing of co-migrating unlabeled marker RNA (see Materials and methods). The position of the three-residue product (lanes 4 and 11) is identical to the position of CpApC formed by the dinucleotide-primed reaction (Goodrich and Tjian, 1994; Holstege et al., 1996). (B) Initiation complexes were assembled on the AdML+15G promoter as in (A). The reaction analyzed in lanes 1–5 received 60 μM ATP and 30 μM [α-32P]CTP, and reactions of lanes 6–11 received 60 μM ATP and 1 μM [α-32P]CTP. It should be noted that the specific activity of [α-32P]CTP is 30 times higher in lanes 6–11 than in lanes 1–5. Reactions were stopped 0.5 (lane 6), 1 (lanes 1 and 7), 2 (lanes 2 and 8), 4 (lanes 3 and 9), 8 (lanes 4 and 10) or 16 min (lanes 5 and 11) after the addition of nucleotides. The position of the ApC product is indicated on the left. The arrows indicate the larger RNA products. Download figure Download PowerPoint To investigate further the phenomenon of read-through by pol II, we tested the effects of CTP concentration and of incubation time on the formation of RNA products. Figure 3B shows that multiple RNA products can be detected when the CTP concentration is increased to 30 μM (lanes 1–5). Synthesis of such products is also dependent on a prolonged incubation. In addition, in permanganate sensitivity assays, we observed that increasing the CTP concentration in the assay results in increased sensitivities in position +3 to +8, whereas the −9/−2 region is not affected (data not shown). Together, these observations strongly support the hypothesis that expansion of the open region to position +8 in the presence of nucleotides, which should allow only formation of the first phosphodiester bond, is caused by complexes transcribing beyond register 2. Whereas read-through products are detected easily in its absence, the RNA chain terminator suppresses the formation of RNAs longer than expected. The conclusion of pol II read-through has important implications for the interpretation of earlier studies (including our own) in which it was assumed that nucleotide deprivation stalled the pol II enzyme at a particular register. Even in the presence of a chain terminator, it is important to titrate carefully the concentration of ribonucleotides in the reaction. The RNA products which are shorter than expected may result from abortive transcription. Abortive RNAs are released rapidly from a transcription complex and their formation should not complicate the permanganate analysis. Abortive RNA synthesis by pol II stops in register 11 The possibility of abortive RNA synthesis is supported by the observation in Figure 3A that shorter products are formed in much higher amounts than longer products. It should be noted that the 15 nt product contains six labeled CMP residues, whereas only one CMP is present in the 2 nt product of the AdML+3G reaction (see Figure 1). The primary criterion for abortive RNA synthesis is the accumulation of excess product over the amount of template present in the reaction (Jacob et al., 1994; Gralla, 1996). To investigate whether formation of short RNAs fulfilled this criterion, a time course experiment was performed to measure formation of RNA products from the various AdMLP templates. Figure 4A shows the accumulation of products using 20 fmol of the AdML+3G or +15G templates in the reaction. Whereas the 15G product reaches a plateau very early after nucleotide addition (Figure 4A, lanes 9–15), the amount of both the 3G and 2 products increases linearly over the time of the assay. Product formation on the other AdMLP templates was analyzed similarly, and Figure 4B shows a graphic representation of this analysis. We found that after 1 min, the 11G and 15G products reach 70% of their maximal levels. After 4 min of incubation, no further increase is observed. In contrast, products shorter than 11 nt show a continuous accumulation over time. This is observed most dramatically with the 3G, 4G and 6G products, but the amount of 9G product also shows a linear increase with time. The 10G product represents an intermediate situation. At 15 min, 50% more 10G RNA is formed than 15G product. Quantitation of the products of Figure 4A showed that after 15 min ∼20 fmol of RNA (2 and 3G RNA) was formed in the AdML+3G reaction, which contained 20 fmol of template. Figure 4.Time course analysis of product formation with the different AdMLP templates. (A) Reactions were assembled as described in the legend of Figure 3A using 20 fmol of the AdML+3G (lanes 2–8) or AdML+15G promoter fragments (lanes 9–15). The reaction analyzed in lane 1 received no template DNA. Reactions received 60 μM ATP, 10 μM [α-32P]CTP and 120 μM 3′-OMeGTP (lanes 2–8), or 60 μM ATP, 10 μM [α-32P]CTP, 10 μM UTP and 120 μM 3′-OMeGTP (lanes 1 and 9–15). Reactions were stopped after 0.5 (lanes 2 and 9), 1 (lanes 3 and 10), 2 (lanes 4 and 11), 4 (lanes 5 and 12), 6 (lanes 6 and 13), 10 (lanes 7 and 14) and 15 min (lanes 1, 8 and 15) and RNA products were analyzed as described in Materials and methods. The positions of the products are indicated. (B) This figure is a graphic representation of time course experiments analyzing product formation with different AdMLP templates. Reactions were assembled and processed as described in the legend of Figure 2A. To the right, the AdMLP templates used are indicated. Only single bands were used for quantitation by phosphorimager analysis (except for the AdML+3G fragment which yielded the 2 and 3G bands). The values plotted on the y-axis were derived by dividing the results of each promoter by the number of cytosines incorporated in the RNA product. The values are relative to the amount of product from the AdML+15G fragment at 6 min. The relative amounts of 3G, 4G, 6G, 9G, 10G and 11G product after 15 min are 23.5, 8.0, 7.6, 2.5, 1.5 and 1.0, respectively. Download figure Download PowerPoint In order to show conclusively that abortive transcription is involved in the synthesis of short RNA products, we tested whether less DNA template could be used in the assays. It was found that the minimal amount of AdML+15G template to sustain the level of 15G formation is 4 fmol of DNA fragment in the reaction (data not shown). The experiment in Figure 4B was repeated using 4 fmol of the different AdMLP templates to determine the amount of RNA product formed in 15 min reactions. The AdML+2G template could not be tested since the assay would require [α-32P]3′-OMeGTP, which is not commercially available. Therefore, formation of the 2 nt product was measured by addition of ATP and CTP to the AdML+15G complex. Figure 5 shows that this results in formation of 2.2 molecules of RNA per DNA molecule. The AdML+3G reaction yields four RNA molecules (2 and 3G products) per template molecule. Formation of the 4G and 6G products is also catalytic, whereas one molecule of 9G RNA is produced per AdML+9G template molecule. In contrast, the AdML+11G and +15G templates yield 0.25 molecules of 11G a