Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Nov;77(11):6556–6560. doi: 10.1073/pnas.77.11.6556

Escherichia coli RNA polymerase in vitro mimics simian virus 40 in vivo transcription when the template is viral nucleoprotein.

E B Jakobovits, S Saragosti, M Yaniv, Y Aloni
PMCID: PMC350324  PMID: 6256744

Abstract

We have used a low-salt detergent-free extraction procedure on cells infected with simian virus 40 to obtain viral nucleoprotein late after infection. Addition of EScherichia coli RNA polymerase and ribonucleotide triphosphates to the viral minichromosomes permitted transcription of RNA from viral templates. This synthesis was initiated predominantly within a fragment of DNA spanning 0.67 to 0.76 map unit on the genome. The synthesis from this region proceeded primarily along the "late" strand in a clockwise direction. These results were in contrast to the synthesis obtained with naked viral DNA in which initiation occurred on other regions of the genome and from which transcription proceeded counterclockwise along the early strand. These findings indicate that the nucleoprotein template or factors tightly associated with it may be responsible for site(s) and strand selection in transcription of simian virus 40.

Full text

PDF
6560

Images in this article

6557Image
on p.6557
6558Image
on p.6558
6559Image
on p.6559

Selected References

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

  1. Aloni Y. Biogenesis and characterization of SV40 and polyoma RNAs in productively infected cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):165–178. doi: 10.1101/sqb.1974.039.01.023. [DOI] [PubMed] [Google Scholar]
  2. Aloni Y., Bratosin S., Dhar R., Laub O., Horowitz M., Khoury G. Splicing of SV40 mRNAs: a novel mechanism for the regulation of gene expression in animal cells. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):559–570. doi: 10.1101/sqb.1978.042.01.058. [DOI] [PubMed] [Google Scholar]
  3. Aloni Y., Dhar R., Khoury G. Methylation of nuclear simian virus 40 RNAs. J Virol. 1979 Oct;32(1):52–60. doi: 10.1128/jvi.32.1.52-60.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aloni Y., Winocour E., Sachs L. Characterization of the simian virus 40-specific RNA in virus-yielding and transformed cells. J Mol Biol. 1968 Feb 14;31(3):415–429. doi: 10.1016/0022-2836(68)90418-x. [DOI] [PubMed] [Google Scholar]
  5. Bellard M., Oudet P., Germond J. E., Chambon P. Subunit structure of simian-virus-40 minichromosome. Eur J Biochem. 1976 Nov 15;70(2):543–553. doi: 10.1111/j.1432-1033.1976.tb11046.x. [DOI] [PubMed] [Google Scholar]
  6. Birkenmeier E. H., Brown D. D., Jordan E. A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes. Cell. 1978 Nov;15(3):1077–1086. doi: 10.1016/0092-8674(78)90291-x. [DOI] [PubMed] [Google Scholar]
  7. Bloom K. S., Anderson J. N. Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion. Cell. 1978 Sep;15(1):141–150. doi: 10.1016/0092-8674(78)90090-9. [DOI] [PubMed] [Google Scholar]
  8. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  9. Cremisi C., Pignatti P. F., Croissant O., Yaniv M. Chromatin-like structures in polyoma virus and simian virus 10 lytic cycle. J Virol. 1975 Jan;17(1):204–211. doi: 10.1128/jvi.17.1.204-211.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feldman H. A., Shelton E. R., Wassarman P. M., DePamphilis M. L. Structure of simian virus 40 chromosomes. Appendix. J Mol Biol. 1978 Nov 15;125(4):511–514. doi: 10.1016/0022-2836(78)90313-3. [DOI] [PubMed] [Google Scholar]
  11. Fernandez-Munoz R., Coca-Prados M., Hsu M. T. Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells. J Virol. 1979 Feb;29(2):612–623. doi: 10.1128/jvi.29.2.612-623.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flint S. J., De Pomerai D. I., Chesterton C. J., Butterworth P. H. Template specificity of eucaryotic DNA-dependent RNA polymerases. Effect of DNA structure and integrity. Eur J Biochem. 1974 Mar 1;42(2):567–579. doi: 10.1111/j.1432-1033.1974.tb03372.x. [DOI] [PubMed] [Google Scholar]
  13. Flint S. J., Weintraub H. M. An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes. Cell. 1977 Nov;12(3):783–794. doi: 10.1016/0092-8674(77)90277-x. [DOI] [PubMed] [Google Scholar]
  14. Foe V. E. Modulation of ribosomal RNA synthesis in Oncopeltus fasciatus: an electron microscopic study of the relationship between changes in chromatin structure and transcriptional activity. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):723–740. doi: 10.1101/sqb.1978.042.01.074. [DOI] [PubMed] [Google Scholar]
  15. Franke W. W., Scheer U., Trendelenburg M., Zentgraf H., Spring H. Morphology of transcriptionally active chromatin. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):755–772. doi: 10.1101/sqb.1978.042.01.076. [DOI] [PubMed] [Google Scholar]
  16. Garber E. A., Seidman M. M., Levine A. J. The detection and characterization of multiple forms of SV40 nucleoprotein complexes. Virology. 1978 Oct 15;90(2):305–316. doi: 10.1016/0042-6822(78)90315-x. [DOI] [PubMed] [Google Scholar]
  17. Garel A., Axel R. Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3966–3970. doi: 10.1073/pnas.73.11.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gariglio P., Llopis R., Oudet P., Chambon P. The template of the isolated native simian virus 40 transcriptional complexes is a minichromosome. J Mol Biol. 1979 Jun 15;131(1):75–105. doi: 10.1016/0022-2836(79)90302-4. [DOI] [PubMed] [Google Scholar]
  19. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Giesecke K., Sippel A. E., Nguyen-Huu M. C., Groner B., Hynes N. E., Wurtz T., Schütz G. A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments. Nucleic Acids Res. 1977 Nov;4(11):3943–3958. doi: 10.1093/nar/4.11.3943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gottesfeld J. M., Garrard W. T., Bagi G., Wilson R. F., Bonner J. Partial purification of the template-active fraction of chromatin: a preliminary report. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2193–2197. doi: 10.1073/pnas.71.6.2193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Green M. H., Miller H. I., Hendler S. Isolation of a polyoma-nucleoprotein complex from infected mouse-cell cultures. Proc Natl Acad Sci U S A. 1971 May;68(5):1032–1036. doi: 10.1073/pnas.68.5.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Griffith J. D. Chromatin structure: deduced from a minichromosome. Science. 1975 Mar 28;187(4182):1202–1203. doi: 10.1126/science.187.4182.1202. [DOI] [PubMed] [Google Scholar]
  24. Hale P., Lebowitz J. Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli. J Virol. 1978 Jan;25(1):298–304. doi: 10.1128/jvi.25.1.298-304.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Horowitz M., Laub O., Bratosin S., Aloni Y. Splicing of SV40 late mRNA is a post-transcriptional process. Nature. 1978 Oct 12;275(5680):558–559. doi: 10.1038/275558a0. [DOI] [PubMed] [Google Scholar]
  26. Jackson A. H., Sugden B. Inhibition by -amanitin of simian virus 40-specific ribonucleic acid synthesis in nuclei of infected monkey cells. J Virol. 1972 Nov;10(5):1086–1089. doi: 10.1128/jvi.10.5.1086-1089.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jakobovits E. B., Aloni Y. Isolation and characterization of various forms of simian virus 40 DNA-protein complexes. Virology. 1980 Apr 15;102(1):107–118. doi: 10.1016/0042-6822(80)90074-4. [DOI] [PubMed] [Google Scholar]
  28. Jakobovits E. B., Bratosin S., Aloni Y. A nucleosome-free region in SV40 minichromosomes. Nature. 1980 May 22;285(5762):263–265. doi: 10.1038/285263a0. [DOI] [PubMed] [Google Scholar]
  29. Kaufmann G., Bar-Shavit R., DePamphilis M. L. Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication. Nucleic Acids Res. 1978 Jul;5(7):2535–2545. doi: 10.1093/nar/5.7.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kedinger C., Gniazdowski M., Mandel J. L., Jr, Gissinger F., Chambon P. Alpha-amanitin: a specific inhibitor of one of two DNA-pendent RNA polymerase activities from calf thymus. Biochem Biophys Res Commun. 1970 Jan 6;38(1):165–171. doi: 10.1016/0006-291x(70)91099-5. [DOI] [PubMed] [Google Scholar]
  31. Korn L. J., Brown D. D. Nucleotide sequence of Xenopus borealis oocyte 5S DNA: comparison of sequences that flank several related eucaryotic genes. Cell. 1978 Dec;15(4):1145–1156. doi: 10.1016/0092-8674(78)90042-9. [DOI] [PubMed] [Google Scholar]
  32. Laub O., Aloni Y. Transcription of simian virus 40. VI. SV 40 DNA-RNA polymerase complex isolated from productively infected cells transcribed in vitro. Virology. 1976 Dec;75(2):346–354. doi: 10.1016/0042-6822(76)90033-7. [DOI] [PubMed] [Google Scholar]
  33. Laub O., Bratosin S., Horowitz M., Aloni Y. The initiation of transcription of SV40 DNA at late time after infection. Virology. 1979 Jan 30;92(2):310–323. doi: 10.1016/0042-6822(79)90136-3. [DOI] [PubMed] [Google Scholar]
  34. Laub O., Jakobovits E. B., Aloni Y. 5,6-dichloro-1-beta-ribofuranosylbenzimidazole enhances premature termination of late transcription of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3297–3301. doi: 10.1073/pnas.77.6.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lebowitz P., Stern R., Ghosh P. K., Weissman S. M. Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP. J Virol. 1977 May;22(2):430–445. doi: 10.1128/jvi.22.2.430-445.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lebowitz P., Weissman S. M. Organization and transcription of the simian virus 40 genome. Curr Top Microbiol Immunol. 1979;87:43–172. doi: 10.1007/978-3-642-67344-3_3. [DOI] [PubMed] [Google Scholar]
  37. Lescure B., Dauguet C., Yaniv M. Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites. J Mol Biol. 1978 Sep 5;124(1):87–96. doi: 10.1016/0022-2836(78)90149-3. [DOI] [PubMed] [Google Scholar]
  38. Lescure B., Oudet P., Chambon P., Yaniv M. Transcription of polyoma virus DNA in vitro. Localization of Escherichia coli RNA polymerase initiation sites. J Mol Biol. 1976 Nov;108(1):83–97. doi: 10.1016/s0022-2836(76)80096-4. [DOI] [PubMed] [Google Scholar]
  39. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. McKnight S. L., Bustin M., Miller O. L., Jr Electron microscopic analysis of chromosome metabolism in the Drosophila melanogaster embryo. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):741–754. doi: 10.1101/sqb.1978.042.01.075. [DOI] [PubMed] [Google Scholar]
  42. Saragosti S., Croissant O., Yaniv M. Localization of the binding sites of prokaryotic and eukaryotic RNA polymerases on simian virus 40 DNA. Eur J Biochem. 1980 May;106(1):25–31. doi: 10.1111/j.1432-1033.1980.tb05993.x. [DOI] [PubMed] [Google Scholar]
  43. Saragosti S., Moyne G., Yaniv M. Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell. 1980 May;20(1):65–73. doi: 10.1016/0092-8674(80)90235-4. [DOI] [PubMed] [Google Scholar]
  44. Schmidt O., Mao J. I., Silverman S., Hovemann B., Söll D. Specific transcription of eukaryotic tRNA genes in Xenopus germinal vesicle extracts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4819–4823. doi: 10.1073/pnas.75.10.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Scott W. A., Wigmore D. J. Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases. Cell. 1978 Dec;15(4):1511–1518. doi: 10.1016/0092-8674(78)90073-9. [DOI] [PubMed] [Google Scholar]
  46. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  47. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  48. Su R. T., DePamphilis M. L. In vitro replication of simian virus 40 DNA in a nucleoprotein complex. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3466–3470. doi: 10.1073/pnas.73.10.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sundin O., Varshavsky A. Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome. J Mol Biol. 1979 Aug 15;132(3):535–546. doi: 10.1016/0022-2836(79)90274-2. [DOI] [PubMed] [Google Scholar]
  50. Varshavsky A. J., Bakayev V. V., Chumackov P. M., Georgiev G. P. Minichromosome of simian virus 40: presence of histone HI. Nucleic Acids Res. 1976 Aug;3(8):2101–2113. doi: 10.1093/nar/3.8.2101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Varshavsky A. J., Sundin O. H., Bohn M. J. SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases. Nucleic Acids Res. 1978 Oct;5(10):3469–3477. doi: 10.1093/nar/5.10.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Varshavsky A. J., Sundin O., Bohn M. A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell. 1979 Feb;16(2):453–466. doi: 10.1016/0092-8674(79)90021-7. [DOI] [PubMed] [Google Scholar]
  53. Waldeck W., Föhring B., Chowdhury K., Gruss P., Sauer G. Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5964–5968. doi: 10.1073/pnas.75.12.5964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]
  55. Weil P. A., Segall J., Harris B., Ng S. Y., Roeder R. G. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J Biol Chem. 1979 Jul 10;254(13):6163–6173. [PubMed] [Google Scholar]
  56. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  57. Westphal H. SV40 DNA strand selection by Escherichia coli RNA polymerase. J Mol Biol. 1970 Jun 14;50(2):407–420. doi: 10.1016/0022-2836(70)90201-9. [DOI] [PubMed] [Google Scholar]
  58. White M., Eason R. Nucleoprotein complexes in simian virus 40-infected cells. J Virol. 1971 Oct;8(4):363–371. doi: 10.1128/jvi.8.4.363-371.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wu G. J. Adenovirus DNA-directed transcription of 5.5S RNA in vitro. Proc Natl Acad Sci U S A. 1978 May;75(5):2175–2179. doi: 10.1073/pnas.75.5.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zasloff M., Felsenfeld G. Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates. Biochemistry. 1977 Nov 15;16(23):5135–5145. doi: 10.1021/bi00642a029. [DOI] [PubMed] [Google Scholar]
  61. Zasloff M., Felsenfeld G. Use of mercury-substituted ribonucleoside triphosphates can lead to artefacts in the analysis of in vitro chromatin transcrits. Biochem Biophys Res Commun. 1977 Apr 11;75(3):598–603. doi: 10.1016/0006-291x(77)91514-5. [DOI] [PubMed] [Google Scholar]
  62. de Pomerai D. I., Chesterton C. J., Butterworth P. H. Preparation of chromatin. Variation in the template properties of chromatin dependent on the method of perparation. Eur J Biochem. 1974 Aug 1;46(3):461–471. doi: 10.1111/j.1432-1033.1974.tb03639.x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES