A Kinetic Model for the Early Steps of RNA Synthesis by Human RNA Polymerase II
2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês
10.1074/jbc.m006401200
ISSN1083-351X
AutoresJennifer F. Kugel, James A. Goodrich,
Tópico(s)RNA and protein synthesis mechanisms
ResumoEukaryotic mRNA synthesis is a highly regulated process involving numerous proteins acting in concert with RNA polymerase II to set levels of transcription from individual promoters. The transcription reaction consists of multiple steps beginning with preinitiation complex formation and ending in the production of a full-length primary transcript. We used pre-steady-state approaches to study the steps of human mRNA transcription at the adenovirus major late promoter in a minimalin vitro transcription system. These kinetic studies revealed an early transition in RNA polymerase II transcription, termed escape commitment, that occurs after initiation and prior to promoter escape. Escape commitment is rapid and is characterized by sensitivity to competitor DNA. Upon completion of escape commitment, ternary complexes are resistant to challenge by competitor DNA and slowly proceed forward through promoter escape. Escape commitment is stimulated by transcription factors TFIIE and TFIIH. We measured forward and reverse rate constants for discrete steps in transcription and present a kinetic model for the mechanism of RNA polymerase II transcription that describes five distinct steps (preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation) and clearly shows promoter escape is rate-limiting in this system. Eukaryotic mRNA synthesis is a highly regulated process involving numerous proteins acting in concert with RNA polymerase II to set levels of transcription from individual promoters. The transcription reaction consists of multiple steps beginning with preinitiation complex formation and ending in the production of a full-length primary transcript. We used pre-steady-state approaches to study the steps of human mRNA transcription at the adenovirus major late promoter in a minimalin vitro transcription system. These kinetic studies revealed an early transition in RNA polymerase II transcription, termed escape commitment, that occurs after initiation and prior to promoter escape. Escape commitment is rapid and is characterized by sensitivity to competitor DNA. Upon completion of escape commitment, ternary complexes are resistant to challenge by competitor DNA and slowly proceed forward through promoter escape. Escape commitment is stimulated by transcription factors TFIIE and TFIIH. We measured forward and reverse rate constants for discrete steps in transcription and present a kinetic model for the mechanism of RNA polymerase II transcription that describes five distinct steps (preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation) and clearly shows promoter escape is rate-limiting in this system. transcription factor of RNA polymerase II adenovirus major late promoter adenosine triphosphate or deoxyadenosine triphosphate nucleotides TATA binding protein mutant calf thymus DNA escape-committed complex elongation complex wild type preinitiation complex formation general transcription factors in the minimal system and RNA polymerase II promoter DNA preinitiation complex initiated complex aborted complex Eukaryotic transcription is a multistep process subject to regulation by promoter-specific transcriptional activators and repressors. The general RNA polymerase II transcription machinery consists of greater than 30 protein subunits (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (848) Google Scholar). The RNA polymerase II core enzyme can synthesize RNA from a DNA template, but requires additional general transcription factors for promoter-specific initiation of transcription. The general transcription factors can be classified into two subgroups: the basal transcription factors (TFIIA,1 TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that are thought to function in transcription at all promoters, and the cofactors that mediate transcriptional activation and appear to be more promoter- and/or regulatory protein-specific (as reviewed in Refs. 2Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (350) Google Scholar, 3Hampsey M. Reinberg D. Curr. Opin. Genet. Dev. 1999; 9: 132-139Crossref PubMed Scopus (138) Google Scholar, 4Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Crossref PubMed Scopus (83) Google Scholar). In current models, the transcription reaction consists of multiple steps, including preinitiation complex formation, open complex formation, promoter escape, promoter clearance, transcript elongation, termination, and reinitiation, all of which have the potential to be regulated by promoter-specific transcriptional activators and repressors (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (848) Google Scholar). Biochemical studies have established that the first step in basal (or unregulated) transcription is the binding of TFIID to core promoter sequences. After TFIID binding, RNA polymerase II and the other general transcription factors assemble on the promoter DNA to form stable preinitiation complexes that become open complexes upon melting of the DNA in the region around the transcription start site (5Reinberg D. Horikoshi M. Roeder R.G. J. Biol. Chem. 1987; 262: 3322-3330Abstract Full Text PDF PubMed Google Scholar, 6Buratowski S. Hahn S. Guarente L. Sharp P.A. Cell. 1989; 56: 549-561Abstract Full Text PDF PubMed Scopus (680) Google Scholar, 7Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Abstract Full Text PDF PubMed Google Scholar, 8Jiang Y. Yan M. Gralla J.D. Mol. Cell. Biol. 1996; 16: 1614-1621Crossref PubMed Google Scholar, 9Holstege F.C.P. van der Vliet P.C. Timmers H.T.M. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar). When open complexes are provided with nucleoside triphosphates, transcription is initiated and RNA polymerase II begins a transformation that results in the dissolution of open complexes and the formation of elongation complexes. This period of the transcription reaction minimally includes the steps of initiation and promoter escape. Initiation begins with synthesis of the first phosphodiester bond, and promoter escape is complete by synthesis of a 15-nt RNA at the adenovirus major late promoter (AdMLP) (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar). During these steps numerous protein-protein and protein-DNA contacts established in the open complex must be broken so that RNA polymerase II can begin to move forward. In addition, new protein-protein and protein-DNA contacts must be established to form an elongation complex. It is thought that during early transcription, TFIID remains bound to the promoter while TFIIB, TFIIE, and TFIIH release from initiation complexes as they proceed forward (11Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Crossref PubMed Scopus (264) Google Scholar). TFIIF remains associated with RNA polymerase II in the ternary elongation complex (11Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Crossref PubMed Scopus (264) Google Scholar). Promoter clearance, a step distinct from promoter escape, is complete when RNA polymerase II has moved far enough to allow another round of initiation at the promoter. RNA transcripts are then elongated by RNA polymerase II in a process that can be interrupted by pausing and influenced by multiple accessory factors (12Reines D. Conaway R. Conaway J. Curr. Opin. Cell Biol. 1999; 11: 342-346Crossref PubMed Scopus (69) Google Scholar). Finally, mRNA synthesis terminates and RNA polymerase II releases from the template and is free to participate in another round of preinitiation complex formation. Studies of RNA polymerase II transcription in reconstituted transcription systems have revealed transitions, which occur during early transcription, that are facilitated by general transcription factors, including TFIIH (as reviewed in Ref. 13Fiedler U. Timmers H. Bioessays. 2000; 22: 316-326Crossref PubMed Scopus (17) Google Scholar). TFIIH contains (d)ATP-dependent helicase activity that has been implicated in promoter melting and promoter escape, as well as the suppression of promoter proximal pausing under conditions of low nucleotide concentrations (9Holstege F.C.P. van der Vliet P.C. Timmers H.T.M. EMBO J. 1996; 15: 1666-1677Crossref PubMed Scopus (205) Google Scholar, 14–25). Two transitions were identified using permanganate footprinting to characterize the melted regions of early transcription complexes on the AdMLP on linear DNA templates in a highly purified transcription system (19Holstege F.C.P. Fiedler U. Timmers H.T.M. EMBO J. 1997; 16: 7468-7480Crossref PubMed Scopus (158) Google Scholar). The first transition, which required TFIIE, TFIIH, and (d)ATP, resulted in an extension of the downstream end of the transcription bubble and was complete when a 4-nt RNA was made. The second transition occurred at +11 and resulted in closing of the upstream end of the transcription bubble and opening at the downstream end. Similar conclusions concerning the role of (d)ATP in propagation of the melted region during early transcription were reached in studies of the adenovirus E4 promoter using a nuclear extract (8Jiang Y. Yan M. Gralla J.D. Mol. Cell. Biol. 1996; 16: 1614-1621Crossref PubMed Google Scholar, 18Yan M. Gralla J.D. EMBO J. 1997; 16: 7457-7467Crossref PubMed Scopus (30) Google Scholar, 26Yan M. Gralla J. J. Biol. Chem. 1999; 274: 34819-34824Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), however, the point of the first transition occurred upon synthesis of a 3-nt RNA product. In these studies it was concluded that the TFIIH helicase functions during the early stages of RNA polymerase II transcription. Deciphering the roles that RNA polymerase II itself plays in transitions that occur during early RNA synthesis would be greatly aided by a kinetic model of the molecular mechanisms that govern the RNA polymerase II transcription reaction under conditions where the TFIIH helicase is not required. A reductionist strategy for developing a quantitative kinetic model for human RNA polymerase II transcription is to measure rate constants for discrete steps in the transcription reaction using a minimal transcription system. Additional factors and transcriptional activators can then be added to the minimal system to study their effects on individual rate constants. This is possible because not all of the general transcription factors are required for basal (non-regulated) transcription in vitro. The TFIID complex, consisting of the TATA binding protein (TBP) and multiple associated factors, can be replaced by the single subunit TBP in basal transcription at TATA-containing promoters (27Peterson M.G. Tanese N. Pugh B.F. Tjian R. Science. 1990; 248: 1625-1630Crossref PubMed Scopus (320) Google Scholar, 28Kao C.C. Lieberman P.M. Schmidt M.C. Zhou Q. Pei R. Berk A.J. Science. 1990; 248: 1646-1650Crossref PubMed Scopus (224) Google Scholar). In addition, TFIIE and TFIIH can be omitted during basal transcription if the promoter is contained on a negatively supercoiled DNA template (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 17Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 29Parvin J.D. Timmers H.T.M. Sharp P.A. Cell. 1992; 68: 1135-1144Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 30Tyree C.M. George C.P. Lira-DeVito L.M. Wampler S.L. Dahmus M.E. Zawel L. Kadonaga J.T. Genes Dev. 1993; 7: 1254-1265Crossref PubMed Scopus (117) Google Scholar). It is thought that negative superhelicity facilitates promoter melting and escape in the absence of the TFIIH helicase (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 17Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 31Timmers H.T.M. EMBO J. 1994; 13: 391-399Crossref PubMed Scopus (90) Google Scholar). Using a minimal transcription system, we previously characterized the kinetics of three stages of human RNA polymerase II transcription at the AdMLP (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar): preinitiation complex formation, promoter escape (synthesis of a 15-nt RNA), and transcript elongation (elongation of a 15-nt RNA into a 390-nt RNA). These kinetic studies revealed that synthesis of a 15-nt RNA from preinitiation complexes limited both the rate and extent of basal transcription in a minimal system. TFIIE and TFIIH, in a (d)ATP-dependent manner, significantly increased the fraction of complexes that produced a 15-nt RNA product. We proposed a model in which early transcription branches into at least two pathways: one that results in functional promoter escape and full-length RNA synthesis, and another in which transcription aborts prior to the completion of promoter escape. TFIIH, in a (d)ATP-dependent reaction, stimulates the fraction of functional complexes that successfully escape the promoter, leading to an increase in the amount of RNA produced (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar). These initial studies did not distinguish between initiation and promoter escape nor did they consider other possible steps that may exist during early RNA synthesis. Our observations, however, suggested that the early steps of RNA synthesis are likely to be a key point for regulating levels of transcription in cells and hence warranted further study. Here we have used a highly purified in vitro transcription system to further study the mechanism and kinetics of the early events of RNA synthesis by human RNA polymerase II at the AdMLP. To aid our studies we used competitor DNA that both inhibits an early step in RNA synthesis and limits transcription to a single round. This enabled us to characterize a specific transition that occurs after initiation and commits RNA polymerase II to the subsequent step of promoter escape. Using pre-steady-state approaches, we measured forward and reverse rate constants for distinct steps in basal transcription at the AdMLP. Because the studies described here were performed in a transcription system containing the minimal number of components necessary to obtain site-specific transcription from the AdMLP (TBP, TFIIB, TFIIF, and core RNA polymerase II), the kinetic model for basal transcription that we established represents the most basic pathway for the RNA polymerase II reaction at this promoter. Recombinant (TBP, TFIIB, TFIIE, and TFIIF) and native (TFIIH and RNA polymerase II) human transcription factors were prepared as described previously (Ref. 10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar, and references therein). The +5mt AdMLP was constructed by site-directed mutagenesis with a primer that anneals to the non-template strand of the promoter (5′-AGGGGAAGTGAGTGAGGACGAACG-3′). Calf thymus DNA (ctDNA, Sigma) was subjected to extensive sonication, phenol-chloroform extraction, and ethanol precipitation, and was used at 275 μg/ml. Transcription reactions were performed in buffer A containing 10 mm Tris·HCl (pH 7.9), 10 mm Hepes (pH 8.0), 10% glycerol, 1 mmdithiothreitol, 4 mm MgCl2, 50 mmKCl, 50 μg/ml bovine serum albumin, and 15 units of RNA Guard (Amersham Pharmacia Biotech). Reactions contained the following amounts of transcription factors: 5 ng of TBP, 10 ng of TFIIB, 6 ng of TFIIF, 50 ng of RNA polymerase II, 15 ng of TFIIE-34, 6 ng of TFIIE-56, and ∼14 ng of TFIIH (where indicated). The DNA template (0.8–1.2 nm) was negatively supercoiled plasmid DNA containing the AdMLP core promoter (−53 to +10) fused to a 380-base pair G-less cassette (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Nucleotides were added at final concentrations of 625 μm ATP, 625 μm CTP, 25 μm[α-32P]UTP (5 μCi per reaction), and, when used, 1 mm ApC. A general scheme for the transcription reactions follows, with details and exceptions included in the figures and figure legends. All transcription factor proteins were preincubated in buffer A for 2 min at 30 °C (10 μl per reaction), after which time promoter DNA in buffer A at 30 °C (10 μl per reaction) was added. The incubation continued for 10 min to allow preinitiation complexes to form, after which either limited nucleotides, a complete set of nucleotides, and/or ctDNA was added as indicated in the figures. Transcription was allowed to proceed for 20 min at 30 °C unless otherwise indicated. Reactions were stopped with 100 μl of a stop solution containing 3.1 m ammonium acetate, 10 μg of carrier yeast RNA, and 15 μg of proteinase K. The samples were ethanol-precipitated and resolved by 6% denaturing polyacrylamide gel electrophoresis. The amount of RNA produced perin vitro transcription reaction was quantitated using a Molecular Dynamics PhosphorImager. After subtracting background, PhosphorImager units from full-length RNA produced at each time point were divided by the average PhosphorImager units produced at the longest time points (in the plateau region) to obtain the fractional completion at each time point. For forward rates, these values were plotted and fit to the equation Fc = 1 −e −kt , where Fcis fractional completion at each time point, t is time in seconds, and k is the rate constant. Rate constants for decay rates were determined using the equationFc = e −kt . Observed rate constants for those steps too fast for obtaining data prior to the plateau region were estimated to be greater than the inverse of the fastest time point taken. In previous studies we found that synthesis of a 15-nt RNA from preinitiation complexes limited both the rate and the extent of a single round of transcription by RNA polymerase II at the AdMLP (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar). At this point in the transcription reaction, promoter escape is complete. Given the importance of the early steps of RNA synthesis in limiting the rate and level of transcription, we decided to study the mechanism of early transcription in greater detail using a minimal RNA polymerase II transcription system consisting of TBP, TFIIB, TFIIF, RNA polymerase II, and the AdMLP contained on a negatively supercoiled DNA template. Under these conditions other transcription factors, including TFIIE and TFIIH, are not required for transcription (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 17Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 29Parvin J.D. Timmers H.T.M. Sharp P.A. Cell. 1992; 68: 1135-1144Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 30Tyree C.M. George C.P. Lira-DeVito L.M. Wampler S.L. Dahmus M.E. Zawel L. Kadonaga J.T. Genes Dev. 1993; 7: 1254-1265Crossref PubMed Scopus (117) Google Scholar). In addition, transcription at the AdMLP on a negatively supercoiled DNA template is not dependent on a source of (d)ATP that is hydrolyzable at the β-γ bond (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 31Timmers H.T.M. EMBO J. 1994; 13: 391-399Crossref PubMed Scopus (90) Google Scholar). All this is consistent with the lack of requirement for the TFIIH helicase in transcription when DNA templates are negatively supercoiled. Therefore, studying transcription under these conditions allows us to assess the intrinsic properties of the RNA polymerase II enzyme in the mechanism of early transcription under conditions where an ancillary helicase activity is not required. To facilitate mechanistic studies and limit transcription to a single round we added sonicated calf thymus DNA (ctDNA) to transcription reactions as a competitor. ctDNA sequesters free TBP and RNA polymerase II thereby preventing them from binding the promoter and potentially initiating additional rounds of transcription. Furthermore, adding ctDNA at different time points allows us to isolate individual steps in the transcription reaction by considering only those events that occur either before or after the addition of ctDNA. The effect of ctDNA on transcription is demonstrated in Fig. 1.Lane 1 shows the level of transcription in the absence of ctDNA. Lane 2 demonstrates that ctDNA prevents preinitiation complex formation when added with the AdMLP, prior to the addition of proteins. Lane 3 shows the level of transcription when ctDNA was added with the nucleotides, after preinitiation complexes were allowed to form. Under this latter condition, the amount of transcript decreased approximately 10-fold from that produced in reactions lacking ctDNA (compare lane 1 to lane 3). This 10-fold decrease in transcription could result entirely from ctDNA inhibiting second and later rounds of initiation. Alternatively, all or part of the 10-fold decrease could be caused by ctDNA inhibiting a specific step in the transcription reaction during a single round of RNA synthesis.Figure 1Calf-thymus DNA (ctDNA ) is a useful competitor for kinetic studies of a single round of RNA polymerase II transcription. An overview of the method is shown in the schematic at the top. ctDNA was added with the promoter DNA (lane 2) or with the nucleotides (lane 3). The position of the 390-nt G-less RNA transcript is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether ctDNA inhibits an early step of a single round of RNA synthesis, we developed the method shown in Fig. 2 A. Preinitiation complexes are formed in the absence of nucleotides. After preinitiation complexes form, transcription is initiated by adding a limited set of nucleotides (ApC, UTP, and CTP). Under these conditions RNA polymerase II progresses to +15 and stably pauses at this position due to the lack of ATP in the system. At this point promoter escape is complete, but the promoter is not yet accessible for a second round of initiation. When ATP is added, the pause releases and 15-nt RNAs are elongated into full-length 390-nt G-less transcripts. ctDNA can be added at different points during the staged reaction to ask whether it inhibits any of the aforementioned stages. As diagrammed in Fig. 2 B, we either omitted ctDNA, added ctDNA with the AdMLP as a control (point 1), added ctDNA with the limited nucleotides so it was present during initiation and promoter escape (point 2), or added ctDNA with the ATP after promoter escape had occurred (point 3). Interestingly, there was a 10-fold decrease in the amount of RNA produced when ctDNA was added with the limited nucleotides (lane 3) compared with its addition with ATP (lane 4). Therefore, prior to the completion of promoter escape, 90% of transcription complexes were inhibited by ctDNA and did not produce full-length RNA in the presence of the competitor (compare lane 3 to lane 1). In contrast, ctDNA did not significantly reduce the level of transcription when added with ATP (compare lane 4 to lane 1). This indicates that even in the absence of ctDNA approximately one round of transcription occurred under these conditions. These results suggest that prior to completion of promoter escape a transition occurs in which complexes become resistant to competitor DNA and produce full-length RNA product. We showed above that by the completion of promoter escape a transition occurs in which complexes become resistant to ctDNA. In addition, we previously determined that early steps of RNA synthesis are rate-limiting for a single round of transcription (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar). Therefore, there are three possibilities as to when complexes become resistant to ctDNA: before, during, or after the rate-limiting step. We used the kinetic experiment shown in Fig. 3 A to distinguish between these possibilities. This experiment allowed us to determine how long it took for complexes to become resistant to ctDNA. As shown in Fig. 3 A, ctDNA was added at multiple time points both before and after the addition of limited nucleotides. When added either with or prior to limited nucleotides, a low amount of transcript was produced (lanes 1–3). Within 10 s after the addition of limited nucleotides, however, complexes became resistant to ctDNA, and the amount of transcript produced plateaued at the maximal level (lanes 4–10). These results indicate that a transition occurs within seconds after the addition of nucleotides, during which time a large fraction of complexes become resistant to challenge with ctDNA and produce an RNA product. The finding that complexes rapidly become resistant to ctDNA upon the addition of nucleotides implies that the rate-limiting step occurs subsequent to this transition. To test this, we performed the experiment shown in Fig. 3 B. Preinitiation complexes were given limited nucleotides for 30 s to allow the transition to ctDNA-resistant complexes to occur, after which time ATP and ctDNA were added and the production of full-length product was monitored over time. The results show that the rate of RNA synthesis from these complexes was slow with a rate constant of 2.0 ± 0.4 × 10−3 s−1. This demonstrates that the rate-limiting step of promoter escape occurs after the transition in which complexes became stable to ctDNA. Experiments carried out with ATP, CTP, and UTP gave similar results, thereby confirming that initiating transcription with the dinucleotide as opposed to ATP does not alter promoter escape or the rate at which complexes become resistant to ctDNA (data not shown). These experiments revealed a kinetically distinct step in transcription that occurs within seconds after providing preinitiation complexes with nucleotides and prior to the rate-limiting step of promoter escape, as shown in the model in Fig. 3 C. During this transition, which we term escape commitment, complexes commit to proceeding forward through promoter escape and are characterized by resistance to ctDNA. Escape-committed complexes (RPEC) transform into elongation complexes (RE) during the rate-limiting step of promoter escape, which is complete prior to or at the point of synthesis of a 15-nt RNA. Prior to the completion of escape commitment, in the presence of ctDNA, approximately 90% of preinitiation complexes abort and are likely to be bound by ctDNA (RPA·ctDNA). Only 10% of complexes successfully proceed through promoter escape in the presence of ctDNA. As a result we observed a 10-fold decrease in the level or extent of transcription when ctDNA was included in transcription reactions with the nucleotides. We will refer to the non-functional complexes that are inhibited by ctDNA as "aborted complexes" (RPA), not to be confused with the term "abortive initiation," which refers to the synthesis and release of short RNA products by RNA polymerases. If promoter escape is rate-limiting for RNA polymerase II transcription at the AdMLP, then all steps before and after promoter escape should occur faster than promoter escape. We have previously found that stable preinitiation complex formation and transcript elongation through the G-less cassette are rapid (kPCF > 0.1 s−1 and kE > 0.3 s−1, respectively (10Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar)). Here we have shown that escape commitment is also rapid (kEC > 0.1 s−1) and occurs within seconds of adding nucleotides to preinitiation complexes (Fig. 3 A). These observations led to the prediction that the formation of escape-committed complexes should occur within seconds of combining proteins with promoter DNA and nucleotides. It was formally possible, however, that a slow step existed after preinitiation complex formation and prior to escape commitment that was not detected by previous experiments. To ensure this was not the case and to confirm that all steps prior to promoter escape occur rapidly, we measured the rate at which escape-committed complexes form after combining proteins with promoter DNA and nucleotides. As shown in Fig. 3 D, the significant difference between this experiment and previous experiments is that preinitiation complexes were not allowed to form prior to the addition of limited nucleotides. Therefore each time point encompassed preinitiation complex formation, escape commitment, and all steps in between. Within 15 s after adding AdMLP and limited nucleotides to the proteins, the level of transcription plateaued at the maximum level. Furthermore, the level of transcript observed in this experiment was equal to that observed in reactions lacking ctDNA, thereby confirming that escape commitment had occurred. This demonstrates that all steps prior to and including escape commitment occur rapidly. We hypothesized that escape commitment occurs with the synthesis of a distinct phosphodiester bond during early transcript synthesis. To test whether escape commitment is complete upon formation of 3-nt RNA product, we added ApC and UTP to preinitiation complexes. RNA products 3 nt in length are made abortively when preinitiation complexes at the AdMLP are provided with ApC and UTP (14Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 32Luse D.S. Jacob G.A. J. Biol. Chem. 1987; 262: 14990-14997Abstra
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