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. 1992 May;66(5):3003–3010. doi: 10.1128/jvi.66.5.3003-3010.1992

Characterization of a 7-kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic RNA polymerase II.

B Y Amegadzie 1, B Y Ahn 1, B Moss 1
PMCID: PMC241059  PMID: 1560534

Abstract

A previously unrecognized 7-kDa polypeptide copurified with the DNA-dependent RNA polymerase of vaccinia virus virions. Internal amino acid sequences of the small protein matched a viral genomic open reading frame of 63 codons. Antipeptide antiserum was used to confirm the specific and complete association of the 7-kDa protein with RNA polymerase. The amino acid sequence predicted from the viral gene, named rpo7, was 23% identical to that of the smallest subunit of Saccharomyces cerevisiae RNA polymerase II, and a metal-binding motif, Cys-X-X-Cys-Gly, was located at precisely the same location near the N terminus in the two proteins. RNA analyses demonstrated early transcriptional initiation and termination signals in the rpo7 gene sequence. The viral RNA polymerase subunit was synthesized during the early phase of infection and continued to accumulate during the late phase.

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Selected References

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

  1. Adman E., Watenpaugh K. D., Jensen L. H. NH---S hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4854–4858. doi: 10.1073/pnas.72.12.4854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Ahn B. Y., Rosel J., Cole N. B., Moss B. Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit. J Virol. 1992 Feb;66(2):971–982. doi: 10.1128/jvi.66.2.971-982.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Amegadzie B. Y., Sisler J. R., Moss B. Frame-shift mutations within the vaccinia virus A-type inclusion protein gene. Virology. 1992 Feb;186(2):777–782. doi: 10.1016/0042-6822(92)90046-r. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Broyles S. S., Fesler B. S. Vaccinia virus gene encoding a component of the viral early transcription factor. J Virol. 1990 Apr;64(4):1523–1529. doi: 10.1128/jvi.64.4.1523-1529.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Falkner F. G., Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol. 1988 Jun;62(6):1849–1854. doi: 10.1128/jvi.62.6.1849-1854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Keck J. G., Baldick C. J., Jr, Moss B. Role of DNA replication in vaccinia virus gene expression: a naked template is required for transcription of three late trans-activator genes. Cell. 1990 Jun 1;61(5):801–809. doi: 10.1016/0092-8674(90)90190-p. [DOI] [PubMed] [Google Scholar]
  21. Kolodziej P. A., Woychik N., Liao S. M., Young R. A. RNA polymerase II subunit composition, stoichiometry, and phosphorylation. Mol Cell Biol. 1990 May;10(5):1915–1920. doi: 10.1128/mcb.10.5.1915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mahr A., Roberts B. E. Arrangement of late RNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment. J Virol. 1984 Feb;49(2):510–520. doi: 10.1128/jvi.49.2.510-520.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meis R. J., Condit R. C. Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-beta-thiosemicarbazone (IBT). Virology. 1991 Jun;182(2):442–454. doi: 10.1016/0042-6822(91)90585-y. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  31. Treich I., Riva M., Sentenac A. Zinc-binding subunits of yeast RNA polymerases. J Biol Chem. 1991 Nov 15;266(32):21971–21976. [PubMed] [Google Scholar]
  32. Woychik N. A., Young R. A. RNA polymerase II subunit RPB10 is essential for yeast cell viability. J Biol Chem. 1990 Oct 15;265(29):17816–17819. [PubMed] [Google Scholar]
  33. Wright C. F., Keck J. G., Tsai M. M., Moss B. A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene. J Virol. 1991 Jul;65(7):3715–3720. doi: 10.1128/jvi.65.7.3715-3720.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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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