α-Amanitin Blocks Translocation by Human RNA Polymerase II
2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês
10.1074/jbc.m402163200
ISSN1083-351X
AutoresXue Gong, Yuri A. Nedialkov, Zachary F. Burton,
Tópico(s)Botanical Research and Chemistry
ResumoOur laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using α-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with α-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, α-amanitin inhibits elongation at each translocation step. Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using α-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with α-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, α-amanitin inhibits elongation at each translocation step. “Among those foods that are eaten carelessly, I would place mushrooms. Although mushrooms taste wonderful, they have fallen into disrepute because of a shocking murder. They were the means by which the emperor Tiberius Claudius was poisoned by his wife Agrippina. Thus, she gave the world a poison worse still— her own son Nero” (translation from Latin, Pliny the Elder). The poisoning occurred in the year 54 A.D. Ingestion of the death cap mushroom Amanita phalloides kills humans by destroying the liver and damaging the kidneys (1Wieland T. Science. 1968; 159: 946-952Crossref PubMed Scopus (246) Google Scholar, 2Wieland T. Gotzendorfer C. Zanotti G. Vaisius A.C. Eur. J. Biochem. 1981; 117: 161-164Crossref PubMed Scopus (26) Google Scholar). For an adult, a lethal dose may be as little as 3 g of the more than ample mushroom cap (6–16 cm diameter), and symptoms do not develop until 8–24 h after ingestion. Victims report that the taste of the deadly mushroom does not presage the agony to come. Severe cases require liver and kidney transplant, treatments unavailable to the unfortunate Claudius. The molecular target of α-amanitin is RNAP 1The abbreviations used are: RNAP, RNA polymerase; TF, transcription factor; HDAg, hepatitis δ antigen; EC, elongation complex; HDV, hepatitis δ virus; HBV, hepatitis B virus; C40, 40-nucleotide RNA ending in a 3′-CMP.1The abbreviations used are: RNAP, RNA polymerase; TF, transcription factor; HDAg, hepatitis δ antigen; EC, elongation complex; HDV, hepatitis δ virus; HBV, hepatitis B virus; C40, 40-nucleotide RNA ending in a 3′-CMP. II, for which the toxin is a potent and specific inhibitor (3Lindell T.J. Weinberg F. Morris P.W. Roeder R.G. Rutter W.J. Science. 1970; 170: 447-449Crossref PubMed Scopus (662) Google Scholar). After three decades of study, however, the mode of toxin action has not been resolved. Recently, an x-ray structure was published of α-amanitin soaked into crystals of yeast RNAP II, indicating many of the key atomic contacts that contribute to RNAP II inactivation (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar). Multiple strong interactions between α-amanitin and RNAP II are found in the “funnel,” the “cleft,” and the key “bridge α-helix” regions of the Rpb1 subunit, which constitute the RNAP II “secondary pore” or “pore 1,” a solventaccessible channel into the RNAP II active site. Contacts to the bridge helix are on the opposite face from the RNAP II active site, indicating that α-amanitin is unlikely to block catalysis directly. Bound α-amanitin partially occludes the pore, which has been presumed to be a channel for NTP substrate loading (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar, 5Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (961) Google Scholar, 6Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar) but may be primarily the route of pyrophosphate release (7Nedialkov 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 (71) Google Scholar). Contacts of α-amanitin to the bridge helix are intriguing because bending of the bridge α-helix is thought to drive translocation of the RNA-DNA hybrid (6Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar, 7Nedialkov 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 (71) Google Scholar, 8Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (626) Google Scholar, 9Holmes S.F. Erie D.A. J. Biol. Chem. 2003; 278: 35597-35608Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Translocation is essential during each bond addition cycle to move the RNA-DNA hybrid away from the space that must be cleared to load the next substrate NTP into the active site. Because α-amanitin binds to RNAP II with a relatively straight conformation of the bridge helix and because of key contacts between the bridge helix and α-amanitin, Kornberg and co-workers (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar) suggested that α-amanitin may block translocation by blocking bridge helix bending. Several reports in the literature, however, indicate that RNAP II may be capable of forming multiple phosphodiester bonds without dissociation of α-amanitin (10Chafin D.R. Guo H. Price D.H. J. Biol. Chem. 1995; 270: 19114-19119Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 11Rudd M.D. Luse D.S. J. Biol. Chem. 1996; 271: 21549-21558Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), so α-amanitin must allow occasional translocation without dissociation from RNAP II. Recently, we developed a method for transient state kinetic analysis of RNAP II elongation stimulated by transcription factor IIF (TFIIF) and hepatitis δ antigen (HDAg) (7Nedialkov 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 (71) Google Scholar). After stalling RNAP II briefly by withholding the next substrate NTP, we demonstrated that the EC fractionated into multiple conformational states with distinct elongation kinetics. We and others showed that RNAP II utilizes a branched kinetic mechanism in which the EC partitions between an active synthesis mode and a pausing mode (12Palangat M. Landick R. J. Mol. Biol. 2001; 311: 265-282Crossref PubMed Scopus (77) Google Scholar, 13Nudler E. J. Mol. Biol. 1999; 288: 1-12Crossref PubMed Scopus (97) Google Scholar, 14Erie D.A. Biochim. Biophys. Acta. 2002; 1577: 224-239Crossref PubMed Scopus (56) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The forward synthesis pathway is intricate, involving NTP substrate-induced fit and multiple conformational states, interpreted as distinct translocation states. Notably, our studies strongly suggest that the incoming substrate NTP first pairs with its cognate DNA template base and then induces translocation of the RNA-DNA hybrid, clearing space to seat the incoming dNMP-NTP base pair in the RNAP II active site for chemistry (7Nedialkov 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 (71) Google Scholar). HDAg is a viral RNAP II elongation factor encoded by hepatitis δ virus (HDV) (16Lai M.M. Annu. Rev. Biochem. 1995; 64: 259-286Crossref PubMed Scopus (266) Google Scholar, 17Taylor J.M. Trends Microbiol. 2003; 11: 185-190Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). HDV has an unusual covalently closed circular RNA genome of about 1.7 kilobases in length, which forms a compact, mostly double-stranded RNA rod structure. The HDV rod is maintained and transported as a satellite particle of hepatitis B virus (HBV), and co-infection with HDV and HBV is associated with severe and chronic presentations of human HBV infection. For the purposes of this paper, HDAg was used to facilitate the kinetic analysis of RNAP II elongation so that α-amanitin inhibition could be analyzed more easily (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Yamaguchi 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 (127) Google Scholar). In this work we present the first study in which α-amanitin is added to RNAP II ECs engaged in active RNA synthesis. We employ a “running start” assay in which a large fraction of RNAP II ECs are committed to the active elongation pathway at the time of inhibitor addition (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Funk J.D. Nedialkov Y.A. Xu D. Burton Z.F. J. Biol. Chem. 2002; 277: 46998-47003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Because we utilize rapid quench technology, we can monitor inhibition and elongation with millisecond time resolution, improving the quality of the analysis. Strikingly, we find that the most highly poised conformation of RNAP II ECs is selectively resistant to α-amanitin inhibition. Previously, we identified the most highly poised EC as being posttranslocated, and studies with α-amanitin inhibition appear to confirm this identification. These highly poised ECs are capable of binding NTP substrate and adding a single phosphodiester bond, but they become highly sensitive to α-amanitin inhibition in the subsequent bond addition cycle, indicating that they become incapable of progressing through the next translocation step. 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 previously (20Shapiro D.J. Sharp P.A. Wahli W.W. Keller M.J. DNA (N. Y.). 1988; 7: 47-55Crossref PubMed Scopus (478) Google Scholar). Recombinant HDAg was purified as described by Yamaguchi et al. (18Yamaguchi 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 (127) Google Scholar). Preparation of Elongation Complexes—Detailed protocols for rapid quench-flow experiments have been published previously (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Funk J.D. Nedialkov Y.A. Xu D. Burton Z.F. J. Biol. Chem. 2002; 277: 46998-47003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 21Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar). Briefly, 32P-labeled C40 (40-nucleotide RNA ending in a 3′-CMP) RNAP II ECs were formed on MagneSphere (Promega, Madison, WI) metal bead-immobilized templates. Initiation was from the adenovirus major late promoter, which was amplified by polymerase chain reaction using a 5′-biotinylated upstream primer. Biotinylation allowed immobilization on streptavidin-coated beads. Transcription factors were derived from an extract of HeLa nuclei. Initiation and elongation were in transcription buffer (12 mm HEPES, pH 7.9, 12% v/v glycerol, 60 mm KCl (unless otherwise specified), 0.12 mm EDTA, 0.12 mm EGTA, 1.2 mm dithiothreitol, and 0.003% IGEPAL CA-630 (Sigma)) containing 8 mm MgCl2. All steps were at 25 °C. The adenovirus major late promoter template has a modified downstream sequence (1ACTCTCTTCCCCTTCTCTTTCCTTCTCTTCCCTCTCCTCC40AAAGGCCTTT50). The purpose of the 39-nucleotide CT-cassette is to synthesize C40 with addition of 300 μm adenylyl(3′→5′)cytidine dinucleotide, 10 μm dATP, 5 μCi of [α-32P]CTP/reaction (800 Ci/mmol), and 20 μm UTP, bypassing the requirement for addition of ATP and GTP. C40 ECs are washed with 1% Sarkosyl and 0.5 m KCl buffer to dissociate initiation, elongation, pausing, and termination factors contributed by the HeLa extract and then are re-equilibrated with transcription buffer containing 8 mm MgCl2 and 20 μm CTP and UTP. A functionally saturating concentration of HDAg (77 pmol/reaction) is then added. Subsequent steps are performed using the Kintek rapid chemical quench-flow (RQF-3) instrument (Kintek Corporation, Austin, TX), which allows reactions to be started and stopped with millisecond precision. Running Start Protocol—The running start protocol has been described previously (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Funk J.D. Nedialkov Y.A. Xu D. Burton Z.F. J. Biol. Chem. 2002; 277: 46998-47003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 21Nedialkov Y.A. Gong X.Q. Yamaguchi Y. Handa H. Burton Z.F. Methods Enzymol. 2003; 371: 252-262Crossref PubMed Scopus (11) Google Scholar). Elongation was through the sequence 40CAAAGGCC47. C40 ECs were advanced to A43 by bench top addition of 100 μm (working concentration) ATP for 60 s. The reaction shown in Fig. 1 was done with the Kintek RQF-3 run in pulse-chase mode. A43 ECs were loaded through the left sample port. α-Amanitin (Sigma) was added through the right sample port. 1 mm GTP (working concentration) was added through the quench syringe. The reaction was stopped in the collection tube, which contained 300 μlof0.5 m EDTA. To analyze the data shown in Fig. 1C, all inhibition curves were adjusted to begin at 50% total ECs so that data from different experiments could be compared more easily. For the experiments shown in Fig. 2, the RQF-3 was operated in the standard mode. A43 ECs were loaded through the left sample port. GTP and α-amanitin were combined and loaded through the right sample port. 0.5 m EDTA was added through the quench syringe. ECs were collected with a magnetic particle separator and processed by electrophoresis. Gel bands were quantitated using an Amersham Biosciences PhosphorImager.Fig. 2The A43c (posttranslocated) reaction phase is highly resistant to α-amanitin inhibition.A, reaction protocol. αA, α-amanitin. B and C, kinetics of RNAP II elongation in the presence of 500 μm GTP. B, gel data. C, PhosphorImager quantitation. D and E, kinetics of RNAP II elongation in the presence of 5 μm GTP. D, gel data. E, PhosphorImager quantitation. G44+ % indicates the percent of total ECs extended to G44 or longer positions. α-Amanitin concentrations are given in μm units in C and E according to the key in C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RNAP II transcriptional stalling was induced by withholding the next substrate NTP. Upon stalling, the EC was observed to fractionate between multiple functional states. In kinetic analyses, these states were identified as populations of ECs with different rates of elongation and different responses to NTP substrate. Conformational states were shown not to be an artifact of RNAP II purification but rather to reflect distinct conformations of a dynamic enzyme as it progresses between alternate functional modes (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). One proof of this point was that different collections of elongation factors reallocated the distribution of RNAP II ECs, so treatments after purification determined EC elongation potential. Because the RNAP II EC fractionates into multiple conformational states at a stall point, we reasoned that these states might have different sensitivities to and/or binding affinities for α-amanitin. We therefore developed a reaction protocol in which α-amanitin was added to the stalled EC either at the same time or just before substrate NTPs (Fig. 1). The purpose of this experimental design was to determine the kinetics for establishing inhibition at different α-amanitin concentrations. 32P-Labeled C40 RNAP II ECs were purified on bead-immobilized templates by Sarkosyl and salt washing. To facilitate elongation, a functionally saturating amount of the RNAP II elongation factor HDAg was added. C40 ECs were then advanced to and stalled at A43 for 60 s by addition of 100 μm ATP. During the stall, samples were injected into the rapid quenchflow apparatus. Rapid mixing combined α-amanitin with ECs, and after a variable incubation, reactions were chased for 0.145 s with 1 mm GTP (0.145 s is sufficient for all ECs on the active elongation pathway to complete the G44 bond). ECs that failed to bind α-amanitin or remained active despite binding toxin were extended to G44 or longer products. This experiment supports the existence of three distinct conformations of the RNAP II EC at the A43 stall position (here designated A43a, A43b, and A43c; Fig. 1C) based on their differential sensitivity to α-amanitin. The A43a phase is the most highly sensitive to inhibition. A43a represents about 5% of total ECs and is most easily observed at 1 and 2 μm α-amanitin. The A43b phase EC appears to represent about 30% of total ECs. For the A43b phase, the rate of inhibition increases as the α-amanitin concentration increases (Fig. 1, C and D), showing that the inhibition of A43b ECs is limited by the rate of α-amanitin binding. By contrast, A43c ECs are highly resistant to α-amanitin inhibition, and the rate of their inactivation does not increase between 10 and 50 μm α-amanitin concentration, indicating that an EC conformational change (A43c → A43b) is required for their inhibition. The A43c EC is highly resistant to α-amanitin binding and/or inactivation for G44 synthesis but becomes highly sensitive to inhibition in the subsequent bond transition from G44 → G45. α-Amanitin Fails to Inhibit the Posttranslocated RNAP II EC—To characterize the α-amanitin-resistant A43c EC, we did the experiments shown in Fig. 2, in which increasing α-amanitin concentrations were combined with stalled A43 ECs at the same time as GTP substrate addition. Inhibition of G44 synthesis was observed at both high (500 μm) and low (5 μm) GTP concentrations, and multiple α-amanitin concentrations were tested from 10 μm to1mm. Rates of G44 synthesis were tracked for up to 1 s. As shown previously, in the absence of α-amanitin, rate curves are notably biphasic up to 0.1 s, with a rapid burst phase (A43c) and a slower phase (here designated A43a + A43b) (7Nedialkov 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 (71) Google Scholar, 15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Using the experimental format of Fig. 2, the A43a and A43b conformational states of the RNAP II EC cannot be distinguished as they were in the experiments shown in Fig. 1. As expected, the amplitudes of the A43c and A43a + A43b phases are dependent on the GTP substrate concentration. Previously, the more highly poised A43c phase was interpreted as being posttranslocated, and the less highly poised and more GTP-dependent A43a + A43b phase was interpreted as being pretranslocated (7Nedialkov 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 (71) Google Scholar). We show here that the highly poised A43c reaction phase is resistant to 1 mm α-amanitin, but the A43a + A43b phase is fully sensitive. Because the highly poised A43c phase of the reaction resists 1 mm α-amanitin even at 5 μm GTP (Fig. 2D), we confirm the existence of an A43c EC conformation that is resistant to inhibition. α-Amanitin binds RNAP II with a much higher affinity than NTP substrates (7Nedialkov 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 (71) Google Scholar, 10Chafin D.R. Guo H. Price D.H. J. Biol. Chem. 1995; 270: 19114-19119Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 11Rudd M.D. Luse D.S. J. Biol. Chem. 1996; 271: 21549-21558Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22Cochet-Meilhac M. Chambon P. Biochim. Biophys. Acta. 1974; 353: 160-184Crossref PubMed Scopus (180) Google Scholar). Therefore, at a 200-fold molar excess of α-amanitin to GTP (1 mmversus 5 μm), α-amanitin binds susceptible ECs before GTP, yet A43c ECs remain resistant to inhibition. From this analysis, it appears certain that the pretranslocated A43a + A43b phase of the EC is highly sensitive to α-amanitin inhibition, and the posttranslocated A43c phase is highly resistant. Chafin et al. (10Chafin D.R. Guo H. Price D.H. J. Biol. Chem. 1995; 270: 19114-19119Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and Rudd and Luse (11Rudd M.D. Luse D.S. J. Biol. Chem. 1996; 271: 21549-21558Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) have shown that α-amanitin allows synthesis of multiple phosphodiester bonds without apparent dissociation of the inhibitor, although elongation rates in the presence of α-amanitin are very slow. Because we have not observed very long elongation times, we do not observe escape from α-amanitin inhibition. In the previously reported experiments, slow elongation in the presence of α-amanitin may have occurred when the EC briefly escaped suppression (i.e. by breaking the α-amanitinbridge helix contact), translocated, and became resistant through a single bond addition cycle before inhibition was restored. α-Amanitin does not affect the apparent affinity of substrate NTPs for RNAP II (10Chafin D.R. Guo H. Price D.H. J. Biol. Chem. 1995; 270: 19114-19119Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 11Rudd M.D. Luse D.S. J. Biol. Chem. 1996; 271: 21549-21558Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22Cochet-Meilhac M. Chambon P. Biochim. Biophys. Acta. 1974; 353: 160-184Crossref PubMed Scopus (180) Google Scholar), although α-amanitin does occupy the secondary pore (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar), which is thought by some (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar, 5Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (961) Google Scholar, 6Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar), but not by us (7Nedialkov 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 (71) Google Scholar), to be the primary route of NTP loading. Bushnell et al. (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar) reported an x-ray structure of yeast RNAP II crystals soaked with α-amanitin. Based on interactions of α-amanitin with the Rpb1 bridge α-helix, they suggested that α-amanitin may block translocation, which may rely on bridge helix bending (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar, 6Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar). In our kinetic study, their expectations are fully supported. We find that α-amanitin blocks elongation at the translocation step and that once the RNA-DNA hybrid and DNA template translocate forward, the EC becomes fully resistant to inhibition through addition of a single phosphodiester bond but not through subsequent bonds. In Fig. 3, we present a detailed model of RNAP II elongation in the presence and absence of α-amanitin (7Nedialkov 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 (71) Google Scholar). α-Amanitin allows NTP loading and chemistry, at least for the A43c conformational state, which remains transcriptionally active through a single bond addition cycle and becomes inhibited in the subsequent cycle. Furthermore, pyrophosphate release cannot be the only step blocked by α-amanitin. If the pyrophosphate release step were the only step inhibited, A43a, A43b, and A43c would all be expected to advance to G44 before being inhibited. The 60-s delay at A43 is considered sufficient for all stalled ECs to dissociate pyrophosphate (7Nedialkov 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 (71) Google Scholar). We proposed previously the NTP-driven translocation model to describe the RNAP II elongation mechanism (7Nedialkov 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 (71) Google Scholar). One of the major observations on which our model relied was that at a transcriptional stall point, the EC fractionated into multiple conformations, interpreted as distinct translocation states. Using α-amanitin as a dynamic probe of the RNAP II mechanism, we substantially support our earlier model and observations. We identify three conformational states of the RNAP II EC with different sensitivities to α-amanitin (A43a, A43b, and A43c) (Fig. 1). The kinetic state herein designated A43a was not detected previously because A43a merged with the state here designated A43b (compare Figs. 1 and 2). The A43c EC strongly resists α-amanitin inhibition (Figs. 1 and 2). We conclude that our previous designation of A43c as the posttranslocated EC is correct and that the posttranslocated state is resistant to α-amanitin inactivation (Fig. 3). The RNAP II Secondary Pore as an Allosteric Site Controlling Transcription—The secondary pore of multisubunit RNAPs appears to be a major allosteric site for the regulation of transcription elongation (23Artsimovitch I. Chu C. Lynch A.S. Landick R. Science. 2003; 302: 650-654Crossref PubMed Scopus (87) Google Scholar), pausing, arrest, RNA cleavage, and transcriptional restart. α-Amanitin penetrates the pore (4Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (234) Google Scholar) to regulate translocation and to arrest elongation at or before the translocation step. Microcin J25 is an inhibitor of bacterial RNAPs (24Wilson K.A. Kalkum M. Ottesen J. Yuzenkova J. Chait B.T. Landick R. Muir T. Severinov K. Darst S.A. J. Am. Chem. Soc. 2003; 125: 12475-12483Crossref PubMed Scopus (198) Google Scholar, 25Rosengren K.J. Clark R.J. Daly N.L. Goransson U. Jones A. Craik D.J. J. Am. Chem. Soc. 2003; 125: 12464-12474Crossref PubMed Scopus (216) Google Scholar, 26Bayro M.J. Mukhopadhyay J. Swapna G.V. Huang J.Y. Ma L.C. Sineva E. Dawson P.E. Montelione G.T. Ebright R.H. J. Am. Chem. Soc. 2003; 125: 12382-12383Crossref PubMed Scopus (179) Google Scholar, 27Yuzenkova J. Delgado M. Nechaev S. Savalia D. Epshtein V. Artsimovitch I. Mooney R.A. Landick R. Farias R.N. Salomon R. Severinov K. J. Biol. Chem. 2002; 277: 50867-50875Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) that bears functional and structural resemblance to α-amanitin (Fig. 4). 2J. Mukhopadhyay, E. Sineva, and R. Ebright, personal communication. Like α-amanitin, Microcin J25 penetrates the secondary pore of its cognate RNAP to inhibit initiation and elongation. Both α-amanitin and Microcin J25 contain cyclic octapeptide structures. α-Amanitin has a 6-hydroxy-Trp4–Cys8 covalent cross-bridge. The C-terminal peptide tail of Microcin J25 (21 amino acids) forms a lariat protoknot through an N-terminal octapeptide covalent ring (Gly1–Glu8) in imitation of the α-amanitin 6-hydroxy-Trp4–Cys8 cross-bridge. α-Amanitin residues hydroxy-Pro2, -Gly5, and -Gly7 and 6-hydroxy-Trp4 appear to correspond to Microcin J25 Pro7, Gly2, Gly4, and Tyr20. Other peptides that penetrate the RNAP pore include the eukaryotic RNA cleavage and restart factors TFIIS/SII (28Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) and the analogous prokaryotic Gre factors (29Sosunova E. Sosunov V. Kozlov M. Nikiforov V. Goldfarb A. Mustaev A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15469-15474Crossref PubMed Scopus (104) Google Scholar, 30Laptenko O. Lee J. Lomakin I. Borukhov S. EMBO J. 2003; 22: 6322-6334Crossref PubMed Scopus (155) Google Scholar). TFIIS/SII was recently shown to be an allosteric effector of RNAP II pausing, separate from its role in RNA cleavage and transcriptional restart (15Zhang C. Yan H. Burton Z.F. J. Biol. Chem. 2003; 278: 50101-50111Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). A newly discovered class of bacterial antibiotics, the CBR703 series, also penetrates the secondary pore of bacterial RNAP and displays allosteric inhibitory effects on transcription (23Artsimovitch I. Chu C. Lynch A.S. Landick R. Science. 2003; 302: 650-654Crossref PubMed Scopus (87) Google Scholar). We thank Y. Yamaguchi for the HDAg clone. We gratefully acknowledge R. Ebright for sharing information prior to publication.
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