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. 1992 Feb;66(2):971–982. doi: 10.1128/jvi.66.2.971-982.1992

Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit.

B Y Ahn 1, J Rosel 1, N B Cole 1, B Moss 1
PMCID: PMC240799  PMID: 1731116

Abstract

The vaccinia virus DNA-dependent RNA polymerase subunit gene rpo19 was identified, and its expression was examined at RNA and protein levels. Antibody to the multisubunit RNA polymerase purified from virions reacted with a polypeptide with an apparent Mr of 21,000 that was synthesized in reticulocyte lysates programmed with (i) mRNA from infected cells that was isolated by hybridization to DNA subclones of the viral genomic HindIII A fragment and (ii) mRNA made in vitro by transcription of the viral open reading frame A6R. Polyclonal antiserum, raised to a recombinant protein product of the A6R open reading frame which could encode an 18,996-Da protein with an acidic N terminus, reacted with Mr-21,000 and -22,000 polypeptides that cosedimented with purified RNA polymerase. Internal sequencing of the two polypeptides confirmed that both were encoded by A6R, and the gene was named rpo19 to indicate the predicted molecular mass of the polypeptide in kilodaltons. Immunoblotting and metabolic labeling of infected cell proteins indicated that synthesis of the Mr-21,000 polypeptide started early and continued throughout virus infection, whereas the Mr-22,000 form appeared late following DNA replication. RNA analyses suggested that the rpo19 mRNA was expressed from a dual early/late promoter and that the protein-coding region of the mRNA was directly preceded by a short 5' poly(A) leader, apparently initiated within the TAAATG motif at the beginning of the open reading frame.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahn B. Y., Gershon P. D., Jones E. V., Moss B. Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor. Mol Cell Biol. 1990 Oct;10(10):5433–5441. doi: 10.1128/mcb.10.10.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahn B. Y., Jones E. V., Moss B. Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript. J Virol. 1990 Jun;64(6):3019–3024. doi: 10.1128/jvi.64.6.3019-3024.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ahn B. Y., Moss B. Capped poly(A) leaders of variable lengths at the 5' ends of vaccinia virus late mRNAs. J Virol. 1989 Jan;63(1):226–232. doi: 10.1128/jvi.63.1.226-232.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Amegadzie B. Y., Ahn B. Y., Moss B. Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase. J Biol Chem. 1991 Jul 25;266(21):13712–13718. [PubMed] [Google Scholar]
  6. Amegadzie B. Y., Holmes M. H., Cole N. B., Jones E. V., Earl P. L., Moss B. Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase. Virology. 1991 Jan;180(1):88–98. doi: 10.1016/0042-6822(91)90012-z. [DOI] [PubMed] [Google Scholar]
  7. Baroudy B. M., Moss B. Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions. J Biol Chem. 1980 May 10;255(9):4372–4380. [PubMed] [Google Scholar]
  8. Bertholet C., Stocco P., Van Meir E., Wittek R. Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. EMBO J. 1986 Aug;5(8):1951–1957. doi: 10.1002/j.1460-2075.1986.tb04449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bertholet C., Van Meir E., ten Heggeler-Bordier B., Wittek R. Vaccinia virus produces late mRNAs by discontinuous synthesis. Cell. 1987 Jul 17;50(2):153–162. doi: 10.1016/0092-8674(87)90211-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boone R. F., Parr R. P., Moss B. Intermolecular duplexes formed from polyadenylylated vaccinia virus RNA. J Virol. 1979 Apr;30(1):365–374. doi: 10.1128/jvi.30.1.365-374.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Broyles S. S., Moss B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A. 1986 May;83(10):3141–3145. doi: 10.1073/pnas.83.10.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Broyles S. S., Pennington M. J. Vaccinia virus gene encoding a 30-kilodalton subunit of the viral DNA-dependent RNA polymerase. J Virol. 1990 Nov;64(11):5376–5382. doi: 10.1128/jvi.64.11.5376-5382.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Broyles S. S., Yuen L., Shuman S., Moss B. Purification of a factor required for transcription of vaccinia virus early genes. J Biol Chem. 1988 Aug 5;263(22):10754–10760. [PubMed] [Google Scholar]
  14. Cherry J. R., Johnson T. R., Dollard C., Shuster J. R., Denis C. L. Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADR1. Cell. 1989 Feb 10;56(3):409–419. doi: 10.1016/0092-8674(89)90244-4. [DOI] [PubMed] [Google Scholar]
  15. Cooper J. A., Moss B. In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells. Virology. 1979 Jul 30;96(2):368–380. doi: 10.1016/0042-6822(79)90095-3. [DOI] [PubMed] [Google Scholar]
  16. Davison A. J., Moss B. Structure of vaccinia virus early promoters. J Mol Biol. 1989 Dec 20;210(4):749–769. doi: 10.1016/0022-2836(89)90107-1. [DOI] [PubMed] [Google Scholar]
  17. Davison A. J., Moss B. Structure of vaccinia virus late promoters. J Mol Biol. 1989 Dec 20;210(4):771–784. doi: 10.1016/0022-2836(89)90108-3. [DOI] [PubMed] [Google Scholar]
  18. Ensinger M. J. Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase. J Virol. 1987 Jun;61(6):1842–1850. doi: 10.1128/jvi.61.6.1842-1850.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gershon P. D., Moss B. Early transcription factor subunits are encoded by vaccinia virus late genes. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4401–4405. doi: 10.1073/pnas.87.11.4401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247-66, 517-63. doi: 10.1016/0042-6822(90)90294-2. [DOI] [PubMed] [Google Scholar]
  21. Hooda-Dhingra U., Thompson C. L., Condit R. C. Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. J Virol. 1989 Feb;63(2):714–729. doi: 10.1128/jvi.63.2.714-729.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hänggi M., Bannwarth W., Stunnenberg H. G. Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J. 1986 May;5(5):1071–1076. doi: 10.1002/j.1460-2075.1986.tb04324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ink B. S., Pickup D. J. Vaccinia virus directs the synthesis of early mRNAs containing 5' poly(A) sequences. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1536–1540. doi: 10.1073/pnas.87.4.1536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. JOKLIK W. K. The preparation and characteristics of highly purified radioactively labelled poxvirus. Biochim Biophys Acta. 1962 Aug 20;61:290–301. doi: 10.1016/0926-6550(62)90091-9. [DOI] [PubMed] [Google Scholar]
  25. Jones E. V., Moss B. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA. J Virol. 1984 Jan;49(1):72–77. doi: 10.1128/jvi.49.1.72-77.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jones E. V., Puckett C., Moss B. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome. J Virol. 1987 Jun;61(6):1765–1771. doi: 10.1128/jvi.61.6.1765-1771.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kleiman J. H., Moss B. Purification of a protein kinase and two phosphate acceptor proteins from vaccinia virions. J Biol Chem. 1975 Apr 10;250(7):2420–2429. [PubMed] [Google Scholar]
  28. Mosrin C., Riva M., Beltrame M., Cassar E., Sentenac A., Thuriaux P. The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail. Mol Cell Biol. 1990 Sep;10(9):4737–4743. doi: 10.1128/mcb.10.9.4737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moss B., Ahn B. Y., Amegadzie B., Gershon P. D., Keck J. G. Cytoplasmic transcription system encoded by vaccinia virus. J Biol Chem. 1991 Jan 25;266(3):1355–1358. [PubMed] [Google Scholar]
  30. Moss B. Regulation of vaccinia virus transcription. Annu Rev Biochem. 1990;59:661–688. doi: 10.1146/annurev.bi.59.070190.003305. [DOI] [PubMed] [Google Scholar]
  31. Nielsen D. A., Shapiro D. J. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. doi: 10.1093/nar/14.14.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Patel D. D., Pickup D. J. Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5'-terminal poly(A) sequences. EMBO J. 1987 Dec 1;6(12):3787–3794. doi: 10.1002/j.1460-2075.1987.tb02714.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Patel D. D., Pickup D. J. The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases. J Virol. 1989 Mar;63(3):1076–1086. doi: 10.1128/jvi.63.3.1076-1086.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Quick S. D., Broyles S. S. Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase. Virology. 1990 Oct;178(2):603–605. doi: 10.1016/0042-6822(90)90362-u. [DOI] [PubMed] [Google Scholar]
  36. Rosel J. L., Earl P. L., Weir J. P., Moss B. Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. J Virol. 1986 Nov;60(2):436–449. doi: 10.1128/jvi.60.2.436-449.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rosel J., Moss B. Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b. J Virol. 1985 Dec;56(3):830–838. doi: 10.1128/jvi.56.3.830-838.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  39. Schwer B., Visca P., Vos J. C., Stunnenberg H. G. Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader. Cell. 1987 Jul 17;50(2):163–169. doi: 10.1016/0092-8674(87)90212-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thompson C. L., Hooda-Dhingra U., Condit R. C. Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. J Virol. 1989 Feb;63(2):705–713. doi: 10.1128/jvi.63.2.705-713.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van Meir E., Wittek R. Fine structure of the vaccinia virus gene encoding the precursor of the major core protein 4 a. Arch Virol. 1988;102(1-2):19–27. doi: 10.1007/BF01315559. [DOI] [PubMed] [Google Scholar]
  43. Woychik N. A., Young R. A. RNA polymerase II: subunit structure and function. Trends Biochem Sci. 1990 Sep;15(9):347–351. doi: 10.1016/0968-0004(90)90074-l. [DOI] [PubMed] [Google Scholar]
  44. Wright C. F., Moss B. In vitro synthesis of vaccinia virus late mRNA containing a 5' poly(A) leader sequence. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8883–8887. doi: 10.1073/pnas.84.24.8883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yuen L., Moss B. Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6417–6421. doi: 10.1073/pnas.84.18.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]

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