Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307590200
ISSN1083-351X
AutoresChunfen Zhang, Honggao Yan, Zachary F. Burton,
Tópico(s)RNA Interference and Gene Delivery
ResumoWhen RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis δ antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pre-translocated state. When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis δ antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pre-translocated state. Our laboratory has applied transient state kinetic analysis (1.Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (516) Google Scholar, 2.Johnson K.A. Enzymes. 1992; 20: 1-61Crossref Scopus (384) Google Scholar, 3.Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (198) Google Scholar) to probe the mechanism and regulation of elongation by human RNA polymerase II (RNAP II) 1The abbreviations used are: RNAP IIRNA polymerase IIECelongation complexHDAghepatitis δ antigenTFIIFtranscription factor IIFTFIIS/SIItranscription factor, RNAP II, stimulatory/stimulatory factor IIHDVhepatitis δ virus. (4.Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar, 5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In the current work, we concentrate on control of the branch point between the active synthesis pathway and the pausing pathway. We measure the rate by which RNAP II leaves the active synthesis pathway to enter the pausing pathway after encountering a barrier to elongation caused by withholding the next substrate nucleoside triphosphate. RNA polymerase II elongation complex hepatitis δ antigen transcription factor IIF transcription factor, RNAP II, stimulatory/stimulatory factor II hepatitis δ virus. It has been suggested that transcription factor IIF (TFIIF) stimulates RNAP II elongation by suppressing transcriptional pausing (6.Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (127) Google Scholar, 7.Izban M.G. Luse D.S. J. Biol. Chem. 1992; 267: 13647-13655Abstract Full Text PDF PubMed Google Scholar, 8.Tan S. Aso T. Conaway R.C. Conaway J.W. J. Biol. Chem. 1994; 269: 25684-25691Abstract Full Text PDF PubMed Google Scholar). TFIIF is a general initiation and elongation factor for RNAP II, composed of RAP74 and RAP30 subunits (RAP for RNA polymerase II-associating protein). In vitro, TFIIF stimulates elongation about 5–10-fold, achieving rates on chromatin-free DNA templates that are similar to rates observed on chromatinized templates in vivo (6.Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (127) Google Scholar, 7.Izban M.G. Luse D.S. J. Biol. Chem. 1992; 267: 13647-13655Abstract Full Text PDF PubMed Google Scholar, 8.Tan S. Aso T. Conaway R.C. Conaway J.W. J. Biol. Chem. 1994; 269: 25684-25691Abstract Full Text PDF PubMed Google Scholar, 9.Ren D. Lei L. Burton Z.F. Mol. Cell. Biol. 1999; 19: 7377-7387Crossref PubMed Scopus (23) Google Scholar, 10.Lei L. Ren D. Burton Z.F. Mol. Cell. Biol. 1999; 19: 8372-8382Crossref PubMed Scopus (24) Google Scholar, 11.Funk 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, 12.Renner 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 (149) Google Scholar). Based on transient state kinetic studies, our laboratory recently proposed a mechanism for RNAP II elongation stimulated by TFIIF and hepatitis δ antigen (HDAg) (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We have also demonstrated the major defects of the RAP74 I176A mutant in general and transient state elongation assays, providing insight into the functions of TFIIF in the RNAP II mechanism (11.Funk 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). We find that TFIIF exerts a global effect on elongation. TFIIF helps commit elongation complexes (ECs) to the forward synthesis pathway. TFIIF stimulates the rate of chemistry and accelerates a slow step after chemistry in the normal processive transition between bonds, a step that includes translocation and pyrophosphate release (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11.Funk 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). We conjecture that TFIIF binds to an external surface of RNAP II, tightens the RNAP II clamp, which grasps the RNA-DNA hybrid, and optimizes conditions for chemistry during elongation. Furthermore, in the presence of TFIIF, RNAP II utilizes the incoming NTP substrate to drive translocation of the RNA-DNA hybrid past the RNAP II active site (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The small RNA genome of hepatitis δ virus (HDV) is packaged and mobilized as a satellite particle by hepatitis B virus (13.Lai M.M. Annu. Rev. Biochem. 1995; 64: 259-286Crossref PubMed Scopus (269) Google Scholar, 14.Taylor J.M. Curr. Top Microbiol. Immunol. 1999; 239: 107-122PubMed Google Scholar, 15.Taylor J.M. Trends Microbiol. 2003; 11: 185-190Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). HDAg is the sole gene product of HDV. HDAg participates in HDV replication, in part by stimulating ribozyme activities of HDV RNA (16.Jeng K.S. Su P.Y. Lai M.M. J. Virol. 1996; 70: 4205-4209Crossref PubMed Google Scholar, 17.Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Recently, however, HDAg was shown to be a potent stimulator of elongation by RNAP II (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18.Yamaguchi 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 (133) Google Scholar, 19.Yamaguchi Y. Delehouzee S. Handa H. Microbes Infect. 2002; 4: 1169-1175Crossref PubMed Scopus (21) Google Scholar). In the viral life cycle, HDAg is thought to stimulate HDV replication and transcription by helping to convert cellular RNAP II from a DNA template-directed RNAP to an RNA template-directed RNAP that can recognize the HDV genome (20.Fu T.B. Taylor J. J. Virol. 1993; 67: 6965-6972Crossref PubMed Google Scholar, 21.Modahl L.E. Macnaughton T.B. Zhu N. Johnson D.L. Lai M.M. Mol. Cell. Biol. 2000; 20: 6030-6039Crossref PubMed Scopus (107) Google Scholar, 22.Chang J. Taylor J. EMBO J. 2002; 21: 157-164Crossref PubMed Scopus (52) Google Scholar). Because HDV replication and transcription are not yet fully recapitulated using in vitro systems (18.Yamaguchi 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 (133) Google Scholar, 23.Filipovska J. Konarska M.M. RNA (New York). 2000; 6: 41-54Crossref PubMed Scopus (77) Google Scholar), our laboratory has pursued studies of HDAg stimulation of RNAP II using DNA templates (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18.Yamaguchi 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 (133) Google Scholar). In the presence of HDAg, the RNAP II elongation mechanism is significantly altered (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Compared with TFIIF, HDAg reduces the NTP dependence of a slow step that occurs after chemistry. We speculate that this slow step represents a taut → relaxed conformational change in RNAP II that loosens contact with the newly polymerized RNA base and facilitates subsequent release of pyrophosphate. This conformational change might be attributed to a loosening of the RNAP II clamp, and HDAg may regulate the tightness of clamp closure (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Loosening the clamp could contribute to the switch in specificity required for RNAP II to replicate and transcribe the HDV RNA template. In the TFIIF-stimulated mechanism the taut → relaxed transition after chemistry is coupled to NTP-driven translocation. In the HDAg-stimulated mechanism, the taut → relaxed transition does not depend on translocation and is instead followed by NTP-driven translocation. TFIIF and HDAg, therefore, stimulate the same basic RNAP II mechanism but regulate the order of key events (translocation and relaxation of the active site), perhaps by regulating the extent and quality of clamp closure. TFIIS/SII is a general elongation factor known to rescue stalled and arrested ECs (24.Reines D. Ghanouni P. Li Q.Q. Mote Jr., J. J. Biol. Chem. 1992; 267: 15516-15522Abstract Full Text PDF PubMed Google Scholar, 25.Reines D. J. Biol. Chem. 1992; 267: 3795-3800Abstract Full Text PDF PubMed Google Scholar, 26.Wang D. Hawley D.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 843-847Crossref PubMed Scopus (89) Google Scholar, 27.Izban M.G. Luse D.S. Genes Dev. 1992; 6: 1342-1356Crossref PubMed Scopus (235) Google Scholar, 28.Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar, 29.Wind M. Reines D. BioEssays. 2000; 22: 327-336Crossref PubMed Scopus (170) Google Scholar). SII is a functional analogue of eubacterial Gre transcription factors (30.Koulich D. Orlova M. Malhotra A. Sali A. Darst S.A. Borukhov S. J. Biol. Chem. 1997; 272: 7201-7210Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 31.Kulish D. Lee J. Lomakin I. Nowicka B. Das A. Darst S. Normet K. Borukhov S. J. Biol. Chem. 2000; 275: 12789-12798Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). During back-tracking, the RNAP II active site slips off the 3′ end of the RNA chain and scans 3′ → 5′ along the RNA within the RNA-DNA hybrid. Backtracked RNA, which extrudes through the RNAP secondary pore, can be cross-linked to SII and to Gre factors (30.Koulich D. Orlova M. Malhotra A. Sali A. Darst S.A. Borukhov S. J. Biol. Chem. 1997; 272: 7201-7210Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 32.Koulich D. Nikiforov V. Borukhov S. J. Mol. Biol. 1998; 276: 379-389Crossref PubMed Scopus (28) Google Scholar, 33.Powell W. Bartholomew B. Reines D. J. Biol. Chem. 1996; 271: 22301-22304Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). SII stimulates an intrinsic RNA cleavage activity attributed to the RNAP II active site, as indicated by the α-amanitin sensitivity and Mg2+ dependence of the cleavage reaction (28.Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar, 34.Rudd M.D. Izban M.G. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8057-8061Crossref PubMed Scopus (109) Google Scholar, 35.Gu W. Reines D. J. Biol. Chem. 1995; 270: 30441-30447Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Recently, the roles of SII and Gre factors were substantially clarified by x-ray and electron microscopy structural studies demonstrating SII and GreB bound to their respective RNAPs (36.Opalka N. Chlenov M. Chacon P. Rice W.J. Wriggers W. Darst S.A. Cell. 2003; 114: 335-345Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 37.Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). SII and Gre factors penetrate the secondary pore of target RNAPs projecting a bound Mg2+ atom into the RNAP active site. The Mg2+ atom held by SII or GreB is poised to participate with a second Mg2+ atom within the RNAP catalytic center to cleave the nascent RNA chain. SII binding significantly rearranges the conformation of the RNAP II active site and the environment of the RNA-DNA hybrid, indicating that some functions of SII may be mediated by allosteric effects. SII is not observed to strongly stimulate forward synthesis by RNAP II (6.Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (127) Google Scholar, 7.Izban M.G. Luse D.S. J. Biol. Chem. 1992; 267: 13647-13655Abstract Full Text PDF PubMed Google Scholar), so the extent to which SII might participate in forward RNA synthesis is not known. In this paper, we have probed the RNAP II pausing mechanism in the presence of TFIIF, HDAg, and SII, added separately or in combination. We demonstrate combinatorial control of pausing by SII and TFIIF. SII cooperates with TFIIF to merge the RNA synthesis and pausing pathways, resulting in acceleration of rates into and out of the pausing pathway. In contrast, SII and HDAg do not cooperate in this way. Judging from the combinatorial effects of SII and HDAg, the paused EC is unlikely to be backtracked beyond the pretranslocated state, because in the presence of HDAg the paused EC resists SII-mediated transcript cleavage. Under elongation conditions, SII stimulates RNA cleavage primarily in dinucleotide increments (28.Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar, 35.Gu W. Reines D. J. Biol. Chem. 1995; 270: 30441-30447Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 38.Izban M.G. Luse D.S. J. Biol. Chem. 1993; 268: 12874-12885Abstract Full Text PDF PubMed Google Scholar, 39.Izban M.G. Luse D.S. J. Biol. Chem. 1993; 268: 12864-12873Abstract Full Text PDF PubMed Google Scholar). When paused ECs backtrack by a single base from the pretranslocated state, the RNAP II active site is poised for SII-stimulated RNA cleavage, and HDAg may therefore resist cleavage by inhibiting backtracking. Cell Culture, Extracts, and Proteins—HeLa cells were purchased from the National Cell Culture Center (Minneapolis, MN). Extracts of HeLa cell nuclei were prepared as described (40.Shapiro D.J. Sharp P.A. Wahli W.W. Keller M.J. DNA (New York). 1988; 7: 47-55Crossref PubMed Scopus (517) Google Scholar). Recombinant HDAg (18.Yamaguchi 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 (133) Google Scholar) and TFIIF (41.Wang B.Q. Kostrub C.F. Finkelstein A. Burton Z.F. Protein Expression Purif. 1993; 4: 207-214Crossref PubMed Scopus (44) Google Scholar, 42.Wang B.Q. Lei L. Burton Z.F. Protein Expression Purif. 1994; 5: 476-485Crossref PubMed Scopus (39) Google Scholar) were prepared as described. Rapid Quench Elongation Experiments—Detailed protocols for rapid quench-flow experiments have been published previously (4.Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar, 5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11.Funk 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). Briefly, 32P-labeled C40 (40-nucleotide RNA ending in a 3′-CMP) RNAP II ECs were formed on metal bead-immobilized templates in a HeLa transcription extract. Initiation was from the adenovirus major late promoter using a modified downstream sequence (1ACTCTCTTCCCCTTCTCTTTCCTTCTCTTCCCTCTCCTCC40). The purpose of the 39-nucleotide CT cassette was to synthesize C40 with ApC dinucleotide, dATP, [α-32P]CTP, and UTP, bypassing the requirement for ATP and GTP. C40 ECs were washed with 1% Sarkosyl and 0.5 m KCl buffer to dissociate initiation, elongation, pausing, and termination factors, contributed by the HeLa extract, and re-equilibrated with transcription buffer containing 8 mm MgCl2 and 20 μm CTP and UTP. Elongation factors were added as indicated in the experimental protocols. Subsequent steps were performed using the Kintek Rapid Chemical Quench-Flow (RQF-3) instrument. Most experiments were done with the instrument operated in pulse-chase mode. Elongation was through the sequence 40CAAAGGCC47. ATP (added through the right sample port) was added to C40 ECs (left sample port) in a timed pulse (0.01–240 s), followed by a GTP chase, added through the middle syringe (normally the quench syringe). Reactions were quenched as samples were expelled from the RQF-3 into collection tubes containing 0.5 m EDTA. By using the 3-syringe sample mixing chamber, the shortest possible time of the GTP chase was determined by the length of the RQF-3 exit line. We calibrated the shortest chase time to be 0.045 s. Longer chase times were obtained by a programmed delay, holding the sample in the exit line before expulsion into quench solution. The experiment shown in Fig. 6 was done with the RQF-3 run in the standard reaction mode. A 30-s ATP pulse was done on the bench top followed by EC transfer into the left sample port. GTP was added from the right sample port, and samples were quenched after various times, as described (4.Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar, 5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11.Funk 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). Kinetic Modeling—Kinetic models were fit to experimental data using the program KinTekSim (43.Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (682) Google Scholar, 44.Zimmerle C.T. Frieden C. Biochem. J. 1989; 258: 381-387Crossref PubMed Scopus (212) Google Scholar). No ATP- or GTP-dependent steps were considered in these models, because elongation from C40 was found to be largely independent of the ATP concentration, within a large concentration range (10–250 μm ATP), and modeling ATP-dependent steps was not necessary. GTP-dependent effects were also neglected, because high GTP concentrations were analyzed, and GTP chase times were selected that allowed fairly complete conversion of active pathway A43 ECs to G44 and longer products. Model-independent analysis (45.Foster J.E. Holmes S.F. Erie D.A. Cell. 2001; 106: 243-252Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 46.Dunlap C.A. Tsai M.D. Biochemistry. 2002; 41: 11226-11235Crossref PubMed Scopus (103) Google Scholar) was done with the program Microcal Origin version 6.1, fitting rate data to single, double, or triple exponential rate curves, as appropriate. In previous work, we indicated that stalled RNAP II ECs tend to revert to the pretranslocated state (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), consistent with the yeast RNAP II EC structure, which is found primarily in the pretranslocated conformation (47.Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (758) Google Scholar). We also noted that stalling induces transcriptional pausing, because the efficiency of rapid elongation from the stall site was highest with the shortest stall times, showing that the longer the stall time, the more ECs would pause (4.Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar). This trend toward pausing, however, was only observed for a few seconds, after which time a steady state condition was established in which the efficiency of elongation remained constant for several minutes. We interpreted this observation to mean that the pausing and active synthesis pathways could assume a dynamic equilibrium that required 1–10 s to establish but would remain stable for minutes before the EC would enter backtracked and arrested elongation modes. Kinetics of Stalling and Entry into the Paused EC—In the current work, we measure the efficiency of rapid RNAP II elongation from a stall position as a function of stall time. Extending the RNA through the sequence 40CAAAGG45, RNAP II ECs were advanced from C40 to A43, by addition of 200 μm ATP (Fig. 1). After variable times of stalling from 0.25 to 240 s, 250 μm GTP was added, and after a 0.55-s elongation time, reactions were quenched with EDTA. The GTP chase time was selected because 0.55 s was sufficient to extend activated A43 ECs to G44 or longer positions, but the paused A43 ECs were not extended (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11.Funk 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). Because a 0.55-s GTP chase was necessary to sample the distribution of active and paused ECs, the time resolution of this experiment is somewhat limited, but the rates that we measure are slow enough that this is not a severe complication. A further difficulty is that, because of slow escape from C40, not all C40 ECs can be delivered to the A43 position rapidly. A43 and longer products are only detected after about 1 s, so resolution on the time axis is limited to about 1.5 s. By considering only those ECs that reach A43 or longer positions, however, this experimental difficulty was largely overcome. The efficiency of elongation from A43 is defined as G44+/A43+, in which G44+ includes G44 plus all longer transcripts and A43+ includes A43 plus all longer transcripts. By doing the calculation based on efficiency, ECs that do not advance from C40 to A43 are neglected in the analysis and do not compromise the estimation of the equilibration rate. To an extent, this analysis is complicated because A43 ECs are continually forming throughout the course of the reaction, and the stall time is therefore somewhat variable for different A43 ECs. The rates we estimate therefore are likely to be a slight underestimate of the true value, because the dwell time at A43 is slightly shorter than the time of the ATP pulse. There is, however, no better experimental means to measure the rates of equilibration between the activated and the paused state, because the reaction cannot commence with a completely synchronous population of active pathway A43 ECs. Despite these limitations, the observed decrease in elongation efficiency as a function of the ATP pulse time (Fig. 1, B and C) can be described by a single-phase exponential curve and a first-order apparent rate, indicating that the rate for equilibration of the active synthesis and pausing pathways can be estimated by this method. Fig. 1B shows RNAP II elongation in the absence (lanes 1–22) and presence of SII (lanes 23–43). Fig. 1C shows PhosphorImager quantitation of the data shown in Fig. 1B, along with data from two replicate experiments, to estimate the kinetics of pausing. Fitting the data to single exponential curves, the rate of equilibration between the active and pausing pathways was estimated as ke = 0.18 ± 0.03, 0.21 ± 0.03, and 0.19 ± 0.03 s–1 (ke for rate of equilibration; errors are reported as standard error). The rate ke for equilibration of the system should be equal to kp + k–p (rate constants into and out of the paused EC). Values for ke, kp, and k–p for the entire paper are shown in Table I.Table IValues for ke, kp, and k-pFactors-SII+SIIkekpk-pkekpk-ps-1s-1s-1s-1s-1s-1No factor0.18 ± 0.030.21 ± 0.030.140.05>1.72.60.950.19 ± 0.03HDAg0.32 ± 0.020.29 ± 0.020.110.070.64 ± 0.110.330.370.28 ± 0.02TFIIF0.19 ± 0.010.18 ± 0.010.0960.10>14NDaND, not determined.NDaND, not determined.0.21 ± 0.01a ND, not determined. Open table in a new tab As controls, samples were run with an ATP pulse from 1 to 240 s without a GTP chase (Fig. 1B, lanes 2–7). Very little background GMP incorporation is detected, even with a 240-s pulse, indicating that there is little GTP contamination in the ATP used in the pulse and that misincorporation of AMP for GMP is not significant during the time course of the experiment. Effects of TFIIS/SII on Pausing and Transcript Cleavage— Because SII regulates RNAP II arrest and transcript cleavage, an experiment was done with addition of SII to determine whether SII regulates pausing by RNAP II. In the absence of other elongation factors, however, we found that, when preincubated with the RNAP II EC, SII was very aggressive in stimulating backtracking and cleavage of the stalled C40 EC (experiment not shown). The C40 EC was not stable, even though during the preincubation reactions contain 20 μm CTP and UTP, which should allow cleaved ECs to re-extend to the C40 position. In order to compare RNAP II elongation ± SII, SII was added at the same time as ATP, making the time of SII addition variable along with the time of EC stalling at A43. In the experiment shown, the GTP chase time was 0.15 s, but results were similar with a chase time of 0.55 s (not shown). SII is functional in this assay, because it stimulates dinucleotide cleavage from C40→ U38, from U38 to smaller RNAs, and from A43→ A41. SII allows for pausing at A43, because the majority of transcripts that reach A43 do not extend to G44 with a 0.15- or 0.55-s GTP chase time, and these chase times are sufficient for active pathway ECs to advance (5.Nedialkov Y.A. Gong X.Q. Hovde S.L. Yamaguchi Y. Handa H. Geiger J.H. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11.Funk 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). However, the rates in and out of the paused state can no longer be resolved because the efficiency of elongation from A43 is not observed to change with time of stalling at A43 (Fig. 1B (lanes 36–43) and Fig. 1C (open symbols)). Paused and active A43 ECs appear to have equilibrated within at least 2 s, so 2 s might represent about 5× t½ of the rate of equilibration between active synthesis and pausing. Based on this estimate, the rate of equilibration should be faster than 1.7 s–1. Therefore, SII may accelerate rates into and out of the pausing pathway. On the other hand, some of the apparent effect of SII on pausing mi
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