An Isatin-β-thiosemicarbazone-resistant Vaccinia Virus Containing a Mutation in the Second Largest Subunit of the Viral RNA Polymerase Is Defective in Transcription Elongation
2004; Elsevier BV; Volume: 279; Issue: 43 Linguagem: Inglês
10.1074/jbc.m408167200
ISSN1083-351X
AutoresCindy Prins, Steven G. Cresawn, Richard Condit,
Tópico(s)Bacteriophages and microbial interactions
ResumoThe vaccinia virus RNA polymerase is a multi-subunit enzyme that contains eight subunits in the postreplicative form. A prior study of a virus called IBTr90, which contains a mutation in the A24 gene encoding the RPO132 subunit of the RNA polymerase, demonstrated that the mutation results in resistance to the anti-poxvirus drug isatin-β-thiosemicarbazone (IBT). In this study, we utilized an in vitro transcription elongation assay to determine the effect of this mutation on transcription elongation. Both wild type and IBTr90 polymerase complexes were studied with regard to their ability to pause during elongation, their stability in a paused state, their ability to release transcripts, and their elongation rate. We have determined that the IBTr90 complex is specifically defective in elongation compared with the WT complex, pausing longer and more frequently than the WT complex. We have built a homology model of the RPO132 subunit with the yeast pol II rpb2 subunit to propose a structural mechanism for this elongation defect. The vaccinia virus RNA polymerase is a multi-subunit enzyme that contains eight subunits in the postreplicative form. A prior study of a virus called IBTr90, which contains a mutation in the A24 gene encoding the RPO132 subunit of the RNA polymerase, demonstrated that the mutation results in resistance to the anti-poxvirus drug isatin-β-thiosemicarbazone (IBT). In this study, we utilized an in vitro transcription elongation assay to determine the effect of this mutation on transcription elongation. Both wild type and IBTr90 polymerase complexes were studied with regard to their ability to pause during elongation, their stability in a paused state, their ability to release transcripts, and their elongation rate. We have determined that the IBTr90 complex is specifically defective in elongation compared with the WT complex, pausing longer and more frequently than the WT complex. We have built a homology model of the RPO132 subunit with the yeast pol II rpb2 subunit to propose a structural mechanism for this elongation defect. Vaccinia virus, the prototypic Orthopoxvirus, contains ∼200 kb of double-stranded linear DNA. Vaccinia virus replicates in the cytoplasm of host cells and encodes its own multi-subunit RNA polymerase and associated transcription machinery, making it a convenient model for transcriptional studies (1Moss B. Knipe D.M. Howley P.M. 4th Ed. Field's Virology. 2. Lippincott Williams & Wilkins, Philadelphia2001: 2849-2883Google Scholar). There are three temporal stages of transcription after a vaccinia virus infection: early; intermediate; and late. For the purposes of this study, the latter two stages are referred to collectively as postreplicative. Early viral transcription has been well characterized, whereas less is known regarding postreplicative transcription. Each stage of transcription is distinguished from the other stages by the recognition of different promoter classes and the utilization of distinct transcription factors. Both early and postreplicative transcription complexes have eight polymerase subunits in common. Of these, RPO147 and RPO132, the products of the J6R and A24R genes, respectively, have some sequence similarity to the two largest subunits of the yeast pol II RNA polymerase. Another subunit, RPO30, which is the product of the E4L gene, has some sequence similarity to the yeast-positive transcription elongation factor TFIIS (2Ahn B.Y. Gershon P.D. Jones E.V. Moss B. Mol. Cell. Biol. 1990; 10: 5433-5441Crossref PubMed Scopus (75) Google Scholar, 3Broyles S.S. Pennington M.J. J. Virol. 1990; 64: 5376-5382Crossref PubMed Google Scholar). The early vaccinia RNA polymerase contains one additional subunit, the product of the H4L gene, Rap94, which is not present in postreplicative transcription complexes (4Baroudy B.M. Moss B. J. Biol. Chem. 1980; 255: 4372-4380Abstract Full Text PDF PubMed Google Scholar, 5Spencer E. Shuman S. Hurwitz J. J. Biol. Chem. 1980; 255: 5388-5395Abstract Full Text PDF PubMed Google Scholar, 6Ahn B.Y. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3536-3540Crossref PubMed Scopus (74) Google Scholar, 7Deng L. Shuman S. J. Biol. Chem. 1994; 269: 14323-14328Abstract Full Text PDF PubMed Google Scholar, 8Kane E.M. Shuman S. J. Virol. 1993; 67: 2689-2698Crossref PubMed Google Scholar). Rap94 appears to be involved in the recognition of promoter classes, because polymerase complexes that contain Rap94 transcribe only early and not late genes and complexes that do not contain Rap94 transcribe only late and not early genes (6Ahn B.Y. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3536-3540Crossref PubMed Scopus (74) Google Scholar, 7Deng L. Shuman S. J. Biol. Chem. 1994; 269: 14323-14328Abstract Full Text PDF PubMed Google Scholar, 9Ahn B.Y. Gershon P.D. Moss B. J. Biol. Chem. 1994; 269: 7552-7557Abstract Full Text PDF PubMed Google Scholar, 10Wright C.F. Coroneos A.M. J. Virol. 1995; 69: 2602-2604Crossref PubMed Google Scholar). Several factors distinguish early transcription elongation and termination from postreplicative transcription elongation and termination. The elongation complex of early genes is fairly stable in the presence of salt and sarkosyl, and one early transcription elongation factor, nucleoside triphosphate phosphohydrolase I, has been identified (11Broyles S.S. Moss B. J. Virol. 1987; 61: 1738-1742Crossref PubMed Google Scholar, 12Rodriguez J.F. Kahn J.S. Esteban M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9566-9570Crossref PubMed Scopus (36) Google Scholar, 13Deng L. Shuman S. Genes Dev. 1998; 12: 538-546Crossref PubMed Scopus (62) Google Scholar). Early transcripts contain an intrinsic termination signal, U5NU, 30–50 nucleotides (nt) 1The abbreviations used are: nt, nucleotide; pol, polymerase; IBT, isatin-β-thiosemicarbazone; WT, wild type.1The abbreviations used are: nt, nucleotide; pol, polymerase; IBT, isatin-β-thiosemicarbazone; WT, wild type. upstream of the mRNA 3′ end (14Shuman S. Moss B. J. Biol. Chem. 1988; 263: 6220-6225Abstract Full Text PDF PubMed Google Scholar, 15Yuen L. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6417-6421Crossref PubMed Scopus (213) Google Scholar, 16Hagler J. Luo Y. Shuman S. J. Biol. Chem. 1994; 269: 10050-10060Abstract Full Text PDF PubMed Google Scholar). Nucleoside triphosphate phosphohydrolase I and the virus-coded mRNA-capping enzyme are both required for early gene transcription termination, which results in the production of transcripts from any given early gene with 3′ ends that are uniform in length (15Yuen L. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6417-6421Crossref PubMed Scopus (213) Google Scholar, 16Hagler J. Luo Y. Shuman S. J. Biol. Chem. 1994; 269: 10050-10060Abstract Full Text PDF PubMed Google Scholar, 17Deng L. Hagler J. Shuman S. J. Biol. Chem. 1996; 271: 19556-19562Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 18Luo Y. Mao X. Deng L. Cong P. Shuman S. J. Virol. 1995; 69: 3852-3856Crossref PubMed Google Scholar). Postreplicative genes do not respond to the early termination signal and lack any other known termination signal. Their transcripts from any given intermediate or late gene are heterogeneous in size at their 3′ ends (19Cooper J.A. Wittek R. Moss B. J. Virol. 1981; 39: 733-745Crossref PubMed Google Scholar, 20Mahr A. Roberts B.E. J. Virol. 1984; 49: 510-520Crossref PubMed Google Scholar). At least three vaccinia virus-encoded proteins are believed to be involved in the regulation of postreplicative transcription elongation and termination. A postreplicative transcript release factor, A18, has been identified in vivo and characterized in vitro (21Xiang Y. Simpson D.A. Spiegel J. Zhou A. Silverman R.H. Condit R.C. J. Virol. 1998; 72: 7012-7023Crossref PubMed Google Scholar, 22Lackner C.A. Condit R.C. J. Biol. Chem. 2000; 275: 1485-1494Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and two putative postreplicative transcription elongation factors, G2 and J3, have been identified through in vivo experiments (23Latner D.R. Xiang Y. Lewis J.I. Condit J. Condit R.C. Virology. 2000; 269: 345-355Crossref PubMed Scopus (30) Google Scholar, 24Black E.P. Condit R.C. J. Virol. 1996; 70: 47-54Crossref PubMed Google Scholar). To confirm the roles of G2 and J3 as elongation factors, an in vitro elongation assay is essential but first the assay must be utilized to evaluate the basic biochemistry of postreplicative transcription with regard to elongation rate and stability in the presence of salt, sarkosyl, and heparin. The anti-poxvirus drug isatin-β-thiosemicarbazone (IBT) has been useful in vivo for studying postinitiation events in postreplicative gene transcription. IBT has no effect on early transcription, but during postreplicative transcription, IBT causes read-through transcription resulting in the production of longer than wild type (WT) length transcripts, suggesting that the drug affects elongation or termination. Read-through transcription from converging promoters leads to the production of excess double-stranded RNA. This causes the activation of the cellular 2-5A pathway and induction of RNase L activity, which inhibits viral growth (25Cohrs R.J. Condit R.C. Pacha R.F. Thompson C.L. Sharma O.K. J. Virol. 1989; 63: 948-951Crossref PubMed Google Scholar). Both IBT-dependent and IBT-resistant mutants can be isolated, and these mutants provide insight into genes that regulate postreplicative transcription (26Katz E. Margalith E. Winer B. Lazar A. J. Gen. Virol. 1973; 21: 469-475Crossref PubMed Scopus (15) Google Scholar). Mutants that are dependent on IBT map to genes that seem to encode elongation factors, specifically G2 and J3 (23Latner D.R. Xiang Y. Lewis J.I. Condit J. Condit R.C. Virology. 2000; 269: 345-355Crossref PubMed Scopus (30) Google Scholar). IBT-dependent null mutations in these genes have been shown in vivo to produce shorter than WT transcripts in the absence of IBT (23Latner D.R. Xiang Y. Lewis J.I. Condit J. Condit R.C. Virology. 2000; 269: 345-355Crossref PubMed Scopus (30) Google Scholar, 24Black E.P. Condit R.C. J. Virol. 1996; 70: 47-54Crossref PubMed Google Scholar). Therefore, in the absence of IBT, G2 and J3 mutants that are IBT-dependent make postreplicative transcripts that are too short to encode all of the proteins needed to maintain viability of the virus. The addition of IBT to these IBT-dependent viruses causes an increase in the length of postreplicative transcripts that restores viability of the virus. IBT resistance presents a more complicated scenario. The target and precise mechanism of action of IBT are unknown, but IBT resistance could theoretically result from a mutation in a gene needed to activate the drug, a mutation that abrogates the binding of the drug, or a mutation that compensates for the effect of the drug. It has been proposed previously that IBT resistance represents an intermediate phenotype between IBT sensitivity and IBT dependence (27Latner D.R. Thompson J.M. Gershon P.D. Storrs C. Condit R.C. Virology. 2002; 301: 64-80Crossref PubMed Scopus (20) Google Scholar). Specifically, IBT-resistant viruses may produce postreplicative transcripts that are shorter than those produced by an IBT-sensitive virus but not as short as those produced by an IBT-dependent virus. In the absence of IBT, the majority of transcripts would be long enough to encode functional proteins, and in the presence of IBT, the majority of transcripts would not be so long as to form a significant amount of double-stranded RNA. IBT-resistant mutants have been isolated that map to the J3R and A24R genes. The IBT-resistant virus with a mutation in the A24R gene has not been characterized with regard to transcript length in vivo or in vitro (28Condit R.C. Easterly R. Pacha R.F. Fathi Z. Meis R.J. Virology. 1991; 185: 857-861Crossref PubMed Scopus (45) Google Scholar). IBT-resistant viruses with a J3 mutation have recently been studied in vivo with regard to transcript length, and these transcripts do not appear to be measurably different in size from those produced by WT or IBT-sensitive viruses in the absence of IBT or from transcripts produced by IBT-dependent viruses in the presence of IBT (23Latner D.R. Xiang Y. Lewis J.I. Condit J. Condit R.C. Virology. 2000; 269: 345-355Crossref PubMed Scopus (30) Google Scholar). It is possible that subtle size differences in transcript length from IBT-resistant viruses in the presence and absence of IBT were difficult to detect in vivo because of the fact that postreplicative vaccinia virus transcripts are heterogeneous in size. To address the mechanism of IBT action, we have modified an in vitro transcript release assay utilized to identify the A18 protein as a transcript release factor and used it to examine postreplicative transcription elongation of the IBT-resistant virus, IBTr90. The IBTr90 virus containsaTtoC substitution at nt 1384 in the A24 gene, which encodes RPO132, the second largest subunit of the RNA polymerase. Both IBTr90 and WT vaccinia viruses were analyzed with regard to pausing, elongation, and stability of the polymerase complexes in postreplicative transcription. Results demonstrate that in vitro IBTr90 is defective in transcription elongation. Terminology—All of the vaccinia virus strain WR genes discussed in this paper are referred to by their location in a HindIII restriction digest. A new nomenclature based on sequential numbering of the genes has been implemented. These new names are mentioned in the text where appropriate. Cell Culture and Infected Cell Extracts for Transcription—A549 cells were propagated in 1× Dulbecco's modified Eagle's medium with 10% bovine calf serum (Hyclone) at 37 °C with 5% CO2. Confluent 100-mm dishes of A549 cells were infected with either WT vaccinia virus strain WR (29Condit R.C. Motyczka A. Virology. 1981; 113: 224-241Crossref PubMed Scopus (16) Google Scholar) or IBTr90 virus (28Condit R.C. Easterly R. Pacha R.F. Fathi Z. Meis R.J. Virology. 1991; 185: 857-861Crossref PubMed Scopus (45) Google Scholar) at a multiplicity of infection of 15 at 37 °C and then incubated in medium containing 10 mm hydroxyurea, an inhibitor of vaccinia viral DNA replication. The addition of hydroxyurea to infected cells results in extracts that contain only the products of early gene transcription, including factors specific for intermediate transcription. At 16–18 h postinfection, cell extracts were prepared as described previously (24Black E.P. Condit R.C. J. Virol. 1996; 70: 47-54Crossref PubMed Google Scholar). Sequencing—The IBTr90 A24R gene (VACWR144) was PCR-amplified using primers upstream and downstream of the open reading frame. This PCR product was submitted to the University of Florida Interdisciplinary Center for Biotechnology Research for sequencing. Sequencing reads were assembled using the Wisconsin package, version 10.3 (Accelrys Inc., San Diego, CA). Templates—The pG8GU template is a derivative of the pG8G template, which contains the vaccinia virus intermediate G8 promoter upstream of a 375-nt G-less cassette and which was originally derived from the pC2AT19 plasmid (22Lackner C.A. Condit R.C. J. Biol. Chem. 2000; 275: 1485-1494Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 30Sawadogo M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4394-4398Crossref PubMed Scopus (366) Google Scholar). To create pG8GU, the pG8G vector was digested with SacI and BamHI to produce a 328-bp fragment containing the vaccinia intermediate G8 promoter sequence and a 37-nt G-less cassette. This fragment was ligated with a 3188-bp SacI-BamHI fragment from the vector pGEM32F to create a 3526-bp intermediate clone called pG8GI. pG8GI was cleaved with SmaI and BamHI to yield a 3497-bp fragment. Two oligonucleotides (GSI44, 5′-GGGAACAGAGGACGAAAGACGACGCAGACAGGAGCAGCGCTTTTTTTTTCATATGG-3′, and GSI46, 5′-GATCCCATATGAAAAAAAAAGCGCTGCTCCTGTCTGCGTCGTCTTTCGTCCTCTGTTCCC-3′) were annealed to create a 40-bp cassette that was ligated with the 3497-bp SmaI-BamHI vector fragment of pG8GI. The resulting clone, pG8GU, is 3558 nt and contains downstream of the G-less cassette a 40-nt T-less cassette followed by a stretch of 9 Ts. The pfeU1 template is originally derived from the pG8GI template. pG8GI was digested with SmaI and PstI to create a 3483-bp vector fragment. The vaccinia virus F17R and E1L genes were amplified with oligonucleotides GSI9 (5′-CCGGTCCCGGGGATGAATTCTCATTTTGCATCTGC-3′), which anneals at the 5′ end of the F17 coding region and contains a 5′-SmaI site, and GSI27 (5′-CGCGCCTGCAGAGAATTCATATGAATAGGAATCCTGATCAG-3′), which anneals at the 5′ end of the E1L coding region and contains a 3′-PstI site. The resulting PCR product was cut with SmaI and PstI to yield a 1760-bp insert that was ligated with the 3483-bp pG8GI vector fragment. This created the clone pG8Gfe, which contains the vaccinia virus intermediate G8 promoter followed by a 37-nt G-less cassette and the F17R/E1L genes (VACWR056/VACWR057). To create pfeU1, pG8Gfe was digested with BamHI and AccI. This resulted in four fragments; an AccI-AccI fragment of 856 bp containing vector sequences, the G8 promoter, and 550 bp of the F17R/E1L genes; an AccI-BamHI fragment of 359 bp of the F17R/E1L genes; a BamHI-AccI fragment of 599 bp of the F17R/E1L genes; and an AccI-AccI fragment of 3420 bp containing the remaining 233 nt of the F17R/E1L genes and vector DNA. The two AccI-AccI fragments were isolated and ligated together to form the 4276-bp pfeU1. Bead-bound DNA Templates for Transcription—The pG8GU and pfeU1 transcription templates were bound to streptavidin-conjugated Dynabeads M280 (Dynal) as described previously for use in the in vitro transcription assay (22Lackner C.A. Condit R.C. J. Biol. Chem. 2000; 275: 1485-1494Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To bind pG8GU, the plasmid DNA was digested with SacI and HindIII and the 395-bp fragment was isolated from an agarose gel and end-filled with biotin for binding to paramagnetic beads. To bind pfeU1, the plasmid DNA was linearized with SalI and end-filled with biotin for binding to streptavidin-coated paramagnetic beads. In Vitro Transcription Elongation Assay—During the pulse phase of the elongation assay, transcription initiation complexes were formed at the viral G8 promoter by combining 15 μl of infected cell extracts with 5 μl of bead-bound DNA template and 5 μl of transcription buffer for a final volume of 25 μl containing 5 mm MgCl2,25mm HEPES, pH 7.4, 1.6 mm dithiothreitol, 80 mm KOAc, 1 mm ATP, 1 mm UTP, 1 μm CTP, 200 μm 3′-O-methyl-GTP, and 6 μCi of [α-32P]CTP (3000 Ci/mmol stock, PerkinElmer Life Sciences). Complexes were incubated at 30 °C for 20 min to allow elongation to proceed to the end of the 37-nt G-less cassette. Complexes were isolated by binding the paramagnetic bead-bound templates to a magnet and were then washed 3× in 1–1.5 reaction volumes of low salt wash buffer containing 5 mm MgCl2,25mm HEPES, pH 7.4, 1.6 mm dithiothreitol, 80 mm KOAc, 200 μg/μl bovine serum albumin, and 7.5% glycerol. Complexes were resuspended in transcription buffer containing 5 mm MgCl2,25mm HEPES, pH 7.4, 1.6 mm dithiothreitol, 80 mm (pG8GU) or 200 mm (PfeU1) KOAc, 20 units of RNasin (Promega) and were chased in the presence of 1 mm ATP, 1 mm CTP, and 1 mm GTP and varying levels of UTP at 30 °C for the times indicated. To investigate the release of transcripts after the chase reaction, the beads were bound to a magnet and 25 μl of supernatant was removed to a new tube. To each tube, 175 μl of proteinase K mixture (114 mm Tris-HCl, pH 7.5, 14 mm EDTA, 171 mm NaCl, 1.1% SDS, 230 μg/ml proteinase K, and 800 μg of glycogen) was added and reactions were incubated at 37 °C for 30 min. Nucleic acids were extracted with 175 μl of phenol/chloroform and then precipitated by adding 290 μl of 10 m NH4OAc and 400 μl of isopropyl alcohol and incubating at room temperature for 30 min. Samples were centrifuged for 20 min, and the supernatant was removed. Pellets were washed in cold (-20 °C) 70% ethanol and resuspended in 10 μl of formamide loading buffer. Samples were denatured at 95 °C for 2 min and separated on a 10% 8 m urea-PAGE. Gels were fixed, dried, exposed to film, and, where applicable, exposed to a PhosphorImager screen (Amersham Biosciences) that was analyzed using a Storm PhosphorImager and the ImageQuant (Amersham Biosciences) program. Modeling—A homology model of the RPO132 protein was constructed using ClustalW, Swiss-PdbViewer, version 3.7, and the Swiss-Model homology modeling server (swissmodel.expasy.org/) (31Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9507) Google Scholar, 32Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55409) Google Scholar). An amino acid sequence alignment of RPO132 and Saccharomyces cerevisiae rpb2 (Protein Data Bank codes 1i6h and 1nik) was constructed using ClustalW and used by Swiss-PdbViewer to generate a structural alignment. The structural alignment was edited by hand and submitted to the SwissModel as an optimize request for construction of a homology model. The Swiss-PdbViewer was used to export views of the model as Mega-Pov scenes that were rendered using POV-Ray (www.pov-ray.org/), version 3.5, for linux. To investigate vaccinia virus postreplicative transcription elongation, we modified an in vitro release assay that had previously been used to investigate postreplicative termination (22Lackner C.A. Condit R.C. J. Biol. Chem. 2000; 275: 1485-1494Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In this system, a linear DNA template that is biotinylated at one or both ends is bound to streptavidin-coated paramagnetic beads to form a transcription template (Fig. 1). The templates used in this study, pG8GU and pfeU1, are both under the control of the vaccinia virus intermediate G8 promoter followed by a 37-nt G-less cassette in the non-template strand. The DNA template is incubated with vaccinia virus-infected cytoplasmic extracts and with transcription buffer plus ATP, UTP, [32P]CTP, and 3′-O-methyl-GTP to initiate a pulse reaction. The incorporation of the 3′-O-methyl-GTP at the end of the 37-nt G-less cassette causes the polymerase complex to arrest at that point. The template is then isolated by binding the paramagnetic beads to a magnet, and the polymerase complex is washed in transcription buffer to remove any unassociated factors. The bead-bound complexes are resuspended in transcription buffer, and transcription elongation resumes in the chase reaction when NTPs are added and the intrinsic cleavage activity of the RNA polymerase cleaves the 3′-O-methyl-GTP residue from the arrested complex (33Hagler J. Shuman S. J. Biol. Chem. 1993; 268: 2166-2173Abstract Full Text PDF PubMed Google Scholar). This cleavage activity was demonstrated for the early polymerase complex, and the ability of the postreplicative complex to recover from incorporation of a 3′-O-methyl-GTP residue indicates that this cleavage activity is also present at intermediate and late times. The two DNA templates used in this study were designed specifically to evaluate the pausing of the polymerase in the chase reaction. Following the 37-nt G-less cassette, the pG8GU template contains a 40-nt T-less cassette followed by a stretch of 9 T residues in the non-template strand, which is known to induce the pausing of the polymerase complex at limiting UTP chase concentrations (Fig. 1A). This pause site is followed by 41 nt of plasmid DNA. The pfeU1 template contains 603 nt of the vaccinia virus E1L and F17R coding regions downstream of the 37-nt G-less cassette (Fig. 1B). A portion of the E1L/F17R coding region was removed to shift a poly(T) site (15/17 nt are Ts) closer to the G8 promoter on this template. This 17-nt poly(T) site is followed by 233 nt of the remaining E1L/F17R genes and 3320 nt of plasmid DNA. To test the in vitro elongation system in a pausing assay, we initiated a pulse reaction on the pG8GU template using WT vaccinia virus extracts (Fig. 2A, lane 1). The pulse reaction yields heterogeneous transcripts ranging in size from ∼50 to 75 nt due to the presence of a 5′-poly(A) "head" averaging 30 nt in size that is added to postreplicative transcripts as a result of the RNA polymerase complex stuttering at the promoter (34Ahn B.Y. Moss B. J. Virol. 1989; 63: 226-232Crossref PubMed Google Scholar, 35Baldick Jr., C.J. Moss B. J. Virol. 1993; 67: 3515-3527Crossref PubMed Google Scholar, 36Bertholet C. Van Meir E. Heggeler-Bordier B. Wittek R. Cell. 1987; 50: 153-162Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 37Patel D.D. Pickup D.J. EMBO J. 1987; 6: 3787-3794Crossref PubMed Scopus (77) Google Scholar, 38Schwer B. Visca P. Vos J.C. Stunnenberg H.G. Cell. 1987; 50: 163-169Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Transcription complexes were chased in the presence of 1 mm ATP, CTP, and GTP and varying amounts of UTP for 2 or 20 min, and bead-bound transcripts (B) were separated from the released transcripts (R). The percentages of full-length and bound transcripts were calculated and graphed (Fig. 2, B and C). In the absence of UTP, the majority of the transcripts are paused at the poly(T) site in both the 2- and 20-min chase reactions (Fig. 2, A, lanes 2 and 20, and B). As increasing amounts of UTP are added, there is an increase in the amount of full-length products produced. At 1 mm UTP, little pausing seems to occur because the majority of transcripts reach full length only after a 2-min chase (Fig. 2, A, lane 18, and B). As noted previously, the amount of transcript release increases with longer chase times and there is a large increase in the percentage of released transcripts after the 20-min chase compared with the 2-min chase (Fig. 2C) (22Lackner C.A. Condit R.C. J. Biol. Chem. 2000; 275: 1485-1494Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These data indicate that the poly(T) site on the pG8GU template does act as a major pause site for the polymerase complex when the complex is starved for UTP in the chase reaction. Therefore, this template serves as an excellent tool for evaluating the ability of the vaccinia virus RNA polymerase complex to elongate under various conditions. To evaluate transcription elongation on the pfeU1 template, a pulse reaction was initiated on pfeU1 with WT-infected cell extracts. The polymerase complex was washed and chased in the presence of 1 mm ATP, CTP, and GTP and either limiting (10 μm) or saturating (1 mm) amounts of UTP for various times (Fig. 3). An analysis of pause sites at saturating and limiting [UTP] demonstrates sites that are natural pauses on the template and those that are caused by reduced UTP levels, presumably at sites that contain stretches of Ts in the non-template strand of pfeU1. For example, at both UTP concentrations and all of the time points, pausing occurs at 325 and 450 nt, indicating that these are natural pause sites in the vaccinia E1L and F17R genes. Conversely, pausing occurs at 425, 475t, and 600 nt at 10 μm UTP but these pauses do not occur at 1 mm UTP. The 600-nt paused transcript is believed to correspond to the 15/17 poly(T) site in the non-template strand of pfeU1, but it is difficult to match the template sequence precisely to the transcript sizes due to the presence of the poly(A) heads on the vaccinia virus postreplicative transcripts. It is interesting to note that, at limiting amounts of UTP, it takes ∼30 min for the polymerase complex to transcribe to the end of the pfeU1 template (Fig. 3, lane 14) and that the amount of read-through does not appear to increase beyond that time (Fig. 3, compares lanes 14, 15, and 16), although at 60- and 90-min chase times, there is some degradation of larger transcripts (Fig. 3, lanes 15, 16, 31, and 32). The presence of both natural and T-rich pause sites on the pfeU1 template makes it an appropriate tool for investigating transcription of authentic vaccinia virus DNA. To determine the transcription elongation phenotype of an IBT-resistant mutant, we made transcription extracts from cells infected with a mutant vaccinia virus called IBTr90. In vivo, the IBTr90 mutant virus produces plaques of equal size whether grown in the presence or absence of IBT (28Condit R.C. Easterly R. Pacha R.F. Fathi Z. Meis R.J. Virology. 1991; 185: 857-861Crossref PubMed Scopus (45) Google Scholar). This mutant maps to the gene encoding the second largest subunit of the viral RNA polymerase, A24R. Sequencing the IBTr90 A24R gene revealed that it contains a Y462H mutation. We initiated a pulse reaction on the pG8GU template with WT or IBTr90 cytoplasmic extracts, washed, and chased the complexes with 1 mm ATP, CTP, GTP, and varying amounts of UTP for 5 or 30 min (Fig. 4). Overall, there is a decrease of ∼40% in the total amount of transcripts produced in a pulse reaction with the IBTr90 polymerase complex compared with the amount produced by the WT complex (Fig. 4A, compare lanes 1 and 18) (data not shown). There is also a decrease in the percentage of full-length transcripts produced by the IBTr90 polymerase compared with those produced by WT as is shown in the graphs of percentages of full-length transcripts versus chase time, which were calculated for two separate experiments (Fig. 4, A and B). For example, in the WT 5-min chase at 30 μm UTP, roughly half of the transcripts are paused and half of them are full length, whereas in the IBTr90 5-min chase at 30 μm UTP, only 18% transcripts are full length (Fig. 4, A, compare lanes 4 and 21, and B). However, the IBTr90 elongation defect only appears at limiting UTP concentrations (Fig. 4A, compare lanes 3–8 with lanes 20–25 and lanes 11–15 with lanes 28–32, B, and C). At 1 mm UTP in a 5-min chase and at 250 μm and 1 mm UTP in a 30
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