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• W2110473043 abstract "We report a “running start, two-bond” protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis δ antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure. We report a “running start, two-bond” protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis δ antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure. RNA polymerase II elongation complex hepatitis δ antigen transcription factor IIF DNA polymerase Pre-steady state kinetic analysis allows the progress of an enzymatic reaction to be tracked in real time (1Johnson K.A. Enzymes. 1992; 20: 1-61Crossref Scopus (379) Google Scholar, 2Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar), and coupling enzyme functional dynamics to the structure provides the clearest insight into the mechanism. In this paper, we compare the first transient state kinetic studies of human (Homo sapiens) RNAP II1 to the x-ray structure of the yeast (Saccharomyces cerevisiae) RNAP II elongation complex (EC) (3Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (752) Google Scholar). These studies give new insight into the RNAP II mechanism and demonstrate the feasibility of a detailed kinetic study of a highly regulated enzyme that is at the hub of gene control in human cells. There is increasing recognition that transcriptional elongation is highly regulated in eukaryotes (4Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 5Shilatifard A. FASEB J. 1998; 12: 1437-1446Crossref PubMed Scopus (99) Google Scholar, 6Yamaguchi Y. Delehouzee S. Handa H. Microbes Infect. 2002; 4: 1169-1175Crossref PubMed Scopus (21) Google Scholar, 7Kim D.K. Yamaguchi Y. Wada T. Handa H. Mol. Cells. 2001; 11: 267-274PubMed Google Scholar, 8Krogan N.J. Kim M. Ahn S.H. Zhong G. Kobor M.S. Cagney G. Emili A. Shilatifard A. Buratowski S. Greenblatt J.F. Mol. Cell. Biol. 2002; 22: 6979-6992Crossref PubMed Scopus (410) Google Scholar). As an example, hepatitis δ antigen (HDAg) strongly stimulates RNAP II elongation in vitro (6Yamaguchi Y. Delehouzee S. Handa H. Microbes Infect. 2002; 4: 1169-1175Crossref PubMed Scopus (21) Google Scholar, 9Yamaguchi Y. Filipovska J. Yano K. Furuya A. Inukai N. Narita T. Wada T. Sugimoto S. Konarska M.M. Handa H. Science. 2001; 293: 124-127Crossref PubMed Scopus (130) Google Scholar). HDAg is the sole gene product of the small RNA genome of hepatitis δ virus, which is maintained as a satellite particle by hepatitis B virus. The role of HDAg in elongation may be clinically significant because hepatitis δ virus often complicates severe and chronic presentations of human hepatitis B virus infection. The general cellular transcription factor IIF (TFIIF) has been shown to stimulate RNAP II elongation 5–10-fold in vitro, by suppressing transcriptional pausing (10Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Tan S. Aso T. Conaway R.C. Conaway J.W. J. Biol. Chem. 1994; 269: 25684-25691Abstract Full Text PDF PubMed Google Scholar, 12Lei L. Ren D. Burton Z.F. Mol. Cell. Biol. 1999; 19: 8372-8382Crossref PubMed Scopus (24) Google Scholar, 13Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (116) Google Scholar, 14Izban M.G. Luse D.S. J. Biol. Chem. 1992; 267: 13647-13655Abstract Full Text PDF PubMed Google Scholar, 15Price D.H. Sluder A.E. Greenleaf A.L. Mol. Cell. Biol. 1989; 9: 1465-1475Crossref PubMed Scopus (134) Google Scholar, 16Funk J.D. Nedialkov Y.A. Xu D. Burton Z.F. J. Biol. Chem. 2002; 277: 46998-47003Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The role of TFIIF in elongation may be of particular importance during the promoter escape phase of the transcription cycle (17Yan Q. Moreland R.J. Conaway J.W. Conaway R.C. J. Biol. Chem. 1999; 274: 35668-35675Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Dvir A. Biochim. Biophys. Acta. 2002; 1577: 208-223Crossref PubMed Scopus (50) Google Scholar). Here viral HDAg and cellular TFIIF are used as probes of H. sapiens RNAP II elongation. In this work, we use rapid quench kinetics to demonstrate critical NTP-dependent steps during RNA synthesis. First, we analyzed recovery from a stall at a defined template position, in the presence of TFIIF or HDAg. During stall recovery, two fractions of EC were clearly observed on the active pathway, and most significantly, these ECs had different requirements for binding and utilizing the incoming substrate NTP. This observation strongly indicates a substrate NTP-induced fit mechanism, in which the NTP first binds and then helps to convert the EC to a fully active form. Second, in the TFIIF-stimulated mechanism but not in the HDAg-stimulated mechanism, a slow step after phosphodiester bond formation is also highly dependent on the incoming NTP. Thus, in the presence of TFIIF, elongation is NTP-driven at both the beginning and the end of a single bond addition cycle, but only one of these NTPs can be the substrate for phosphodiester bond formation at a single position. The other NTP appears to be the substrate for addition of the subsequent bond. These observations lead to the following conclusions: 1) RNAP II elongates according to a substrate NTP-induced fit mechanism; 2) translocation can be induced by prior NTP binding. Translocation must occur at either the beginning or the end of each bond addition cycle, and in the presence of TFIIF, both are highly dependent on the next templated NTP. Significantly, HDAg eliminates the high NTP dependence of the slow step after phosphodiester bond formation, demonstrating the unusual nature of the RNAP II mechanism in the presence of TFIIF. As with TFIIF, the HDAg-stimulated mechanism shows evidence of substrate NTP- induced fit during escape from a stall, but, unlike TFIIF, NTP dependence is not detected with HDAg in the normal processive transition between bonds. Comparing the kinetics of RNAP II elongation with the yeast EC structure reverses the view of how NTPs are loaded, alters our understanding of the translocation mechanism, and provides new insight into transcriptional efficiency and fidelity. HeLa cells were purchased from the National Cell Culture Center (Minneapolis, MN). Extracts of HeLa cell nuclei were prepared as described (19Shapiro D.J. Sharp P.A. Wahli W.W. Keller M.J. DNA (New York). 1988; 7: 47-55Crossref PubMed Scopus (478) Google Scholar). Recombinant TFIIF (20Wang B.Q. Kostrub C.F. Finkelstein A. Burton Z.F. Protein Expression Purif. 1993; 4: 207-214Crossref PubMed Scopus (44) Google Scholar, 21Wang B.Q. Lei L. Burton Z.F. Protein Expression Purif. 1994; 5: 476-485Crossref PubMed Scopus (39) Google Scholar) and HDAg (9Yamaguchi Y. Filipovska J. Yano K. Furuya A. Inukai N. Narita T. Wada T. Sugimoto S. Konarska M.M. Handa H. Science. 2001; 293: 124-127Crossref PubMed Scopus (130) Google Scholar) were prepared as described. The running start, two-bond elongation assay is shown in Fig. 1 (16Funk J.D. Nedialkov Y.A. Xu D. Burton Z.F. J. Biol. Chem. 2002; 277: 46998-47003Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 23Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol.,. 2003; (in press)Google Scholar). Initiation was from the adenovirus major late promoter with a modified downstream sequence so that a 40-nucleotide transcript can be synthesized in the absence of ATP and GTP. An extract of human HeLa cells was the source of transcription factors. C40 (a 40-nucleotide RNA ending in a 3′-CMP) ECs were synthesized by addition of 10 μm dATP, 300 μm ApC dinucleotide, 5 μCi per reaction [α-32P]CTP, and 20 μm UTP. ECs were then washed with 1% Sarkosyl and 0.5 m KCl, to dissociate initiation, elongation, pausing, and termination factors, and then re-equilibrated with transcription buffer. To commence the reaction, C40 ECs were incubated in transcription buffer containing 8 mm MgCl2 for 10–40 min (the time varies according to the time required for individual sample handling) in the presence of 12 pmol of TFIIF or 77 pmol of HDAg (functionally saturating amounts) and 20 μm (initial working concentration) CTP and UTP (to maintain ECs at C40). Downstream of C40 the sequence is 40CAAAGG45. On the bench top, an equal sample volume of 200 μm ATP (initial working concentration 100 μm) was added in transcription buffer. The ATP pulse time was 60 s with HDAg, 30 s with TFIIF, and 120 s in the absence of an elongation factor. Times were optimized for each procedure (23Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol.,. 2003; (in press)Google Scholar). During the pulse, EC samples were injected into the left sample port of the Kintek Rapid Chemical Quench-Flow (RQF-3) instrument and mixed with GTP added from the right sample port. Due to subsequent equal volume mixing events, the final working NTP concentrations are 5 μm CTP, 5 μm UTP, 50 μm ATP and the indicated concentration of GTP. Rate measurements from 0.002 to 4 s were done using the KinTek instrument, and longer time points were done on the bench top, all at 25 °C. After quenching reactions with 0.5 m EDTA, beads were collected; supernatant was removed, and samples were dissolved in 90% formamide loading dye containing 1% SDS. Samples were boiled for 2 min and RNAs separated in 14–16% polyacrylamide (20:1 acrylamide/bisacrylamide) gels containing 50% w/v urea and 1× Tris borate-EDTA. Gels were analyzed using a Amersham BiosciencesPhosphorImager. Each gel lane was analyzed independently for percent of signal present in G44 plus all longer transcripts or G45 plus all longer transcripts compared with A43 plus all longer transcripts. The data were handled in this way to compensate for occasional inconsistency in recovery or loading of samples. The complex kinetics we report demonstrate multiple conformers of A43 EC at the time of GTP addition in the running start, two-bond protocol. Because ECs were isolated on bead templates from HeLa nuclear extracts, it is reasonable to consider whether A43 ECs differ in their kinetic properties because of experimental treatments or because of damage to a subset of ECs during preparation. However, A43 conformational states are determined by treatments that occur after EC purification. The initial conformational states detected at A43 are different in the presence of TFIIF, HDAg, or in the absence of an elongation factor, showing that protein factors drive RNAP II between functional modes. Furthermore, increasing GTP concentration blurs the distinction between different kinetic states, indicating that RNAP II changes conformation through interactions with substrate, as expected for an RNAP. Also, the distribution of A43 states is dependent on the time of stalling at A43, demonstrating reversibility between states (23Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol.,. 2003; (in press)Google Scholar). A43 conformational states, therefore, are selected based on treatments (protein factors, substrate, and time of incubation) after purification and are not an artifact of preparation. In the purification scheme, RNAP II molecules are selected for the ability to initiate transcription accurately in concert with the general initiation factors, and all C40 and A43 complexes are active in elongation. Sarkosyl and salt treatment appears to strip all contaminating transcription factors and complicating activities from the EC (12Lei L. Ren D. Burton Z.F. Mol. Cell. Biol. 1999; 19: 8372-8382Crossref PubMed Scopus (24) Google Scholar). Kinetic models were designed based on a qualitative assessment of rate data, as described under “Results,” and model independent analysis of rate data (24Foster J.E. Holmes S.F. Erie D.A. Cell. 2001; 106: 243-252Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 25Dunlap C.A. Tsai M.D. Biochemistry. 2002; 41: 11226-11235Crossref PubMed Scopus (98) Google Scholar) (not shown). The program DYNAFIT (26Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1360) Google Scholar), which utilizes non-linear least squares curve fitting to obtain the optimal fit to a kinetic model, was used to estimate rate constants. Most of the rate constants listed in Fig. 4are currently under determined experimentally, so the values reported are meant to represent a simulation of the mechanism with the caveat that future refinement will be necessary to determine fully the accurate rate constant values. The rate constants used for simulations, however, give a reasonable qualitative and quantitative description of the rate equation. The mechanisms we apply fit the primary characteristics of the rate data sets, whereas alternate schemes prove inadequate to model the data. Furthermore, the models we espouse seem consistent with the S. cerevisiae RNAP II EC structure (3Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (752) Google Scholar). The kinetic pathway shown in Fig. 4A is the simplest induced fit mechanism with a pausing pathway that allows access of the active site after an NTP-induced fit conformational change (see under “Results” and “Discussion”). This is an adequate kinetic model for RNAP II elongation stimulated by HDAg. To fit the HDAg-stimulated mechanism requires a minimum of three initial states at A43 as follows: A43a (23% of total ECs); A43b (27% of total ECs); and A43c (50% of total ECs). From model-independent analysis (not shown), the fastest rate is estimated as 1250 s−1, and the fastest rate on the slow pathway (A43a·GTP → A43b·GTP) is >20 s−1. A slow step after chemistry is required to fit the sigmoidal shape of G45 rate curves (Figs. 3B and 4A). The rate of this slow step is well determined as 15.5 ± 0.5 s−1. The pathway shown in Fig. 4B is the simplest induced fit model (including a pausing pathway), with an open active site, that also confers substrate NTP dependence at both the start and end of the G44 bond addition cycle (see under “Results” and “Discussion”). This mechanism is adequate to model rates of elongation stimulated by TFIIF through formation of two phosphodiester bonds. The simple induced fit model (Fig. 4A) is adequate to fit the TFIIF data set through formation of the G44 phosphodiester bond (not shown) but not through formation of the G45 bond. In the presence of TFIIF, a minimum of three initial conformers at A43 are required: A43a (55% of total); A43b (10% of total); and A43c (35% of total). Model independent kinetic analysis indicates that the fastest pathway for G44 synthesis is >500 s−1 (not shown) and is estimated as 1200 s−1 in the model shown in Fig. 4B. The model independent analysis estimates the slower pathway from A43a as >100 s−1 (not shown). Furthermore, there is a requirement for loading two substrate GTPs in a single G44 bond addition cycle. The GTP requirement is fulfilled by loading both the n + 1 andn + 2 GTPs to A43a before translocation. As expected from the S. cerevisiae RNAP II EC structure, GTPs held primarily by base pairing are bound weakly (GTP off-rates of 10,000 s−1; the DYNAFIT program would select faster dissociation rates). Elongation rate data in the presence of TFIIF (Figs.3D and 4B) require a fast elongation pathway that dominates at high GTP concentrations and a slow pathway that dominates at limiting GTP concentrations. Residuals (a statistical test; Fig. 3) indicate that these models converge to experimental data generally within 5 or 10%, indicating the reliability of the simulations. Particularly with HDAg, the curve fits are very close to experimental values (residuals ±5%). The rate constants shown in Fig. 4, A and B, are the best estimates we can offer at this time, although more information is required about individual steps in the mechanism to accurately assign rate constant values, and additional experiments will be required to refine the current models. The rate constants shown in Fig. 4 are constrained to be in approximate thermodynamic balance. The curve fits and residuals shown in Fig. 3 were optimized from the set of rate constants shown in Fig. 4, by relaxing this constraint, so the reported curve fits and residuals represent a slightly better fit to the data set than those obtained from the rate constants shown in Fig. 4. To analyze the mechanism and regulation of human RNA synthesis, we sought a method to obtain transient state kinetic measurements of RNAP II elongation rates through formation of multiple bonds. Precisely stalled RNAP II ECs, immobilized on magnetic beads, were initiated from the adenovirus major late promoter and isolated with Sarkosyl and salt washing (Fig.1). C40 ECs contain a 40-nucleotide,32P-labeled RNA, ending in a 3′-CMP base. The sequence downstream of C40 is40CAAAGG45. Because C40 ECs proved unsuitable for measuring the most rapid elongation rates, a running start, two-bond protocol was adopted. In the presence of 20 μm CTP and UTP, 100 μm ATP was added to advance C40 ECs to the A43 position. After a brief stall at A43, a steady state distribution was established between paused and active A43 ECs (23Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol.,. 2003; (in press)Google Scholar), such that, when GTP was added, rapid rates for G44 and G45 synthesis could be determined reproducibly. In this experimental design, G44 synthesis rates reflect recovery from a stall at A43, and initial G45 synthesis rates reflect processive elongation from G44 → G45, including a translocation of the RNA-DNA hybrid and template DNA. Because the running start assay allowed us to commit a significant fraction of A43 ECs to rapid elongation, we adapted this method to analyze GTP-dependent steps along the forward synthesis path. In Fig. 2, we compare synthesis through the G44 and G45 positions in the presence of the RNAP II elongation factors HDAg and TFIIF. After a brief ATP pulse, GTP was added at the indicated concentrations, and reactions were quenched at various times. The protocol is indicated in Figs. 1 and 2A. The ATP pulse time, optimized for each reaction, is 120 s in the absence of a stimulatory factor (Fig. 2B), 60 s in the presence of HDAg (Fig. 2C), and 30 s in the presence of TFIIF (Fig. 2D). In Fig. 2B, elongation is shown in the absence of a stimulatory factor to confirm that TFIIF and HDAg enhance elongation in the running start assay (compare Fig. 2,B–D). Rates of G44 and G45 synthesis were evaluated in terms of the percent of transcript at G44 or G45 plus all longer transcripts. In this way, rates of disappearance of G44 and G45 could be neglected. The running start, two-bond protocol reveals significant details of the RNAP II elongation mechanism. In the presence of HDAg or TFIIF, synthesis rates for G44 differ from synthesis rates for G45, demonstrating the importance of monitoring two bonds. With the running start method, analysis of the G44 bond is expected to provide detail about RNAP II conformational states and kinetic intermediates and any effects of recovering from the 30- to 120-s stall at A43. Rates of G45 synthesis, on the other hand, are expected to reveal characteristics of the approach to processive elongation. The rates of first G45 appearance should provide insight into translocation, because translocation and pyrophosphate release must occur between synthesis of the G44 and G45 bonds. If only a single bond is tracked, information about translocation could be lost, because the state(s) of translocation prior to addition of substrate cannot be known. In Fig. 3, complete data sets are shown for elongation through the G44 and G45 positions, in the presence of HDAg (Fig. 3, A and B) or TFIIF (Fig. 3, C and D). The comparison between the HDAg- and TFIIF-stimulated data sets provides clear insight into many details of the RNAP II mechanism and its regulation by elongation factors. Curve fits are derived from the kinetic mechanisms shown in Fig. 4,A and B. Residuals shown below each plot in Fig.3 demonstrate how closely experimental data can be fit with these simulations. Analysis of the rates of G44 synthesis (Fig. 3, A andC) indicates that RNAP II must utilize an induced fit mechanism, in which binding substrate GTP modifies the A43 EC conformationally to become catalytically competent. Notably, during the 60- or 30-s stall at A43, delayed ECs fractionate between at least three different A43 states, two of which are evident on the forward synthesis pathway and one of which is strongly paused. Furthermore, two A43 ECs are observed on the active synthesis pathway with differing dependence on the concentration of the incoming GTP substrate. In the presence of HDAg, ECs partition between three states (Fig.3A). Two A43 ECs are found on the active synthesis pathway, but these two EC conformers have different responsiveness to GTP. About 27% of A43 ECs are in the most highly poised state (Fig.3A, fraction b; 0–27% of total ECs;kobs,fast ∼1250 s−1 (see under “Experimental Procedures”)). An additional 23% of A43 ECs are more dependent on GTP substrate to advance rapidly (Fig. 3A,fraction a; 27–50% of total ECs;kobs,slow >20 s−1 (see under “Experimental Procedures”)). Because two classes of A43 EC are detected with different responsiveness to GTP concentration, this is evidence of induced fit in the RNAP II mechanism. GTP binds to a less highly poised EC (fraction a) and converts it to a more highly poised EC (fraction b). In the presence of HDAg, 50% of A43 ECs are initially paused (Fig. 3A; 50–100% of total ECs;kforward ∼0.05 s−1). In the presence of TFIIF, three classes of A43 EC are detected but with different occupancy than those observed with HDAg (compare Fig. 3,A and C). Two of these classes of A43 EC are on the active synthesis pathway, and one is paused. About 10% of A43 ECs elongate rapidly to the G44 position even at GTP concentrations that are too low to support subsequent rapid elongation from G44 to G45 (Fig. 3C, fraction b; 0–10% of total ECs;kobs,fast >500 s−1 (see under “Experimental Procedures”)). These A43 ECs are highly poised to bind GTP substrate and incorporate GMP. A distinct fraction of A43 EC (about 55% of total) elongates rapidly at high GTP concentrations but much more slowly at low GTP concentrations (Fig. 3C,fraction a; 10–65% of total ECs;kobs,slow >100 s−1). This fraction of A43 EC (fraction a) requires prior GTP binding to convert to a catalytically competent state (fraction b), consistent with a substrate GTP-induced fit mechanism for RNAP II elongation. The remaining 35% of A43 ECs (Fig. 3C; 65–100% of total ECs;kforward ∼0.09 s−1) are strongly paused but eventually can be extended (Fig. 2D). Because multiple A43 ECs are detected that respond differently to substrate GTP concentrations, with both HDAg and TFIIF, the RNAP II EC assumes conformations that must first bind GTP and then be converted to a form capable of catalyzing phosphodiester bond formation. Judging from the initial times of G44 and G45 appearance on gels (Fig. 2, B–D), the most rapid rates of G44 synthesis must be 5–10-fold faster than the rate of initial G45 appearance, which is surprisingly slow. If this were not the case, G45 would be detected by the 0.002–0.005-s time points, but G45 is not detected until 0.02–0.05 s, even at high GTP concentration. This conclusion is further demonstrated by quantitation of gel data (Fig. 3, Band D). Analysis of G45 synthesis rate curves shows that the initially slow appearance of G45 can be attributed to a slow conformational step after G44 phosphodiester bond formation. This conclusion is demonstrated by the sigmoidal shapes of the rate curves shown in Fig. 3, B and D. The distinctive shape of these curves can only be described by a slow, normally irreversible step in the RNAP II elongation mechanism after G44 phosphodiester bond synthesis but before rapid G45 synthesis can commence (see Fig. 4,A and B). Note that the interval in which this slow conformational step occurs must include the translocation event between G44 and G45 synthesis. In the presence of HDAg (Fig. 3B), there is a slow step after G44 phosphodiester bond formation that accounts for the slow first appearance of G45. This slow step is indicated by the sigmoidal shapes of G45 synthesis rate curves. Notice that the sigmoidal rate curves all approach the time axis at a similar intersection point (Fig.3B, open arrow). This result shows that, in the presence of HDAg, the lag in G45 first appearance is not highly dependent on GTP concentration, although in the presence of TFIIF (Fig. 3D), the lag duration is highly GTP-dependent. For the HDAg-stimulated mechanism, the slow step after chemistry can be modeled by a first order rate constant, lacking GTP dependence, of 15.5 ± 0.5 s−1 (Fig. 4A). Surprisingly, in the presence of TFIIF, the situation is very different. At 250 and 100 μm GTP, G45 appears in an apparent burst (Fig. 3D), but these rate curves are sigmoidal when plotted with an expanded time axis, indicating the slow step after chemistry (lag of 0.02–0.05 s (Fig. 2D)). From 10 to 25 μm GTP, G45 synthesis rate curves are notably sigmoidal in shape, further demonstrating the slow step after G44 bond formation. Surprisingly, at 1 and 2 μm GTP, almost no G45 synthesis is observed within 5 s, although eventually these ECs will advance (data not shown). This result demonstrates the extreme GTP dependence of this slow step in the TFIIF-stimulated mechanism. So, after a stall at A43 in the presence of TFIIF, both the beginning phase and the ending phase of the G44 bond addition cycle are highly dependent on the next incoming GTP substrates (Fig. 3, C andD). In the running start protocol, this unusual condition arises because elongation was stalled at the A43 position. Because the GTP-driven event at the end of the G44 bond addition cycle occurs after chemistry, this event cannot be attributed to utilization of GTP as a substrate for G44 bond formation and primarily reflects entry of the GTP substrate for G45 synthesis. As with TFIIF, HDAg-mediated recovery from a stall at A43 is GTP-dependent (Fig. 3A), demonstrating the GTP-induced fit mechanism. With HDAg, however, the transition between synthesis of the G44 and G45 bonds is not noticeably dependent on the incoming substrate GTP (Fig. 3B), as it is in the presence of TFIIF (Fig. 3D). Therefore, HDAg and TFIIF regulate the normal processive transition between bonds (translocation) in distinct ways. HDAg simplifies this step and makes it less dependent on GTP. Also, recovery from a stall is found to be a distinct process from the normal processive transition between bonds, because TFIIF and HDAg regulate these steps differently. One result of HDAg-mediated stimulation is that elongation is facilitated at GTP concentrations that are very restrictive in the presence of TFIIF, as if HDAg facilitates a rate-limiting step (translocation) in the RNAP II mechanism (compare Fig. 3, A–D). RNAP II appears to extend an RNA chain according to an induced fit mechanism, in which the active site remains available for substrate NTP binding, even when the conformational change precedes substrate loading. Furthermore, RNAP II establishes a steady state condition between the pausing and active synthesis pathways that is maintained for minutes at the A43 position (23Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol.,. 2003; (in press)Google Scholar). The simplest kinetic model that will satisfy these conditions is shown in Fig. 4A. Induced fit requires that substrate NTP normally binds prior to a conformational step in the mechanism (27Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (" @default.
• W2110473043 created "2016-06-24" @default.
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• W2110473043 date "2003-05-01" @default.
• W2110473043 modified "2023-10-14" @default.
• W2110473043 title "NTP-driven Translocation by Human RNA Polymerase II" @default.
• W2110473043 cites W1000298608 @default.
• W2110473043 cites W1493170168 @default.
• W2110473043 cites W1505354077 @default.
• W2110473043 cites W1533660032 @default.
• W2110473043 cites W1569390933 @default.
• W2110473043 cites W1595863394 @default.
• W2110473043 cites W1813910892 @default.
• W2110473043 cites W1968671224 @default.
• W2110473043 cites W1972061284 @default.
• W2110473043 cites W1978029039 @default.
• W2110473043 cites W1982433583 @default.
• W2110473043 cites W1990083018 @default.
• W2110473043 cites W1995231578 @default.
• W2110473043 cites W2006373999 @default.
• W2110473043 cites W2008940576 @default.
• W2110473043 cites W2028509576 @default.
• W2110473043 cites W2033735656 @default.
• W2110473043 cites W2035438363 @default.
• W2110473043 cites W2036001210 @default.
• W2110473043 cites W2037995065 @default.
• W2110473043 cites W2053312292 @default.
• W2110473043 cites W2053625042 @default.
• W2110473043 cites W2055975305 @default.
• W2110473043 cites W2061228034 @default.
• W2110473043 cites W2064920037 @default.
• W2110473043 cites W2074206760 @default.
• W2110473043 cites W2074507088 @default.
• W2110473043 cites W2076600263 @default.
• W2110473043 cites W2080229694 @default.
• W2110473043 cites W2084622414 @default.
• W2110473043 cites W2086949060 @default.
• W2110473043 cites W2095219787 @default.
• W2110473043 cites W2096260627 @default.
• W2110473043 cites W2096619494 @default.
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• W2110473043 cites W2114997522 @default.
• W2110473043 cites W2128671635 @default.
• W2110473043 cites W2135487357 @default.
• W2110473043 cites W2135646930 @default.
• W2110473043 cites W2141373343 @default.
• W2110473043 cites W2149410625 @default.
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• W2110473043 cites W2153508349 @default.
• W2110473043 cites W2157114285 @default.
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