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. 1995 Sep 11;23(17):3373–3379. doi: 10.1093/nar/23.17.3373

Variable pause positions of RNA polymerase II lie proximal to the c-myc promoter irrespective of transcriptional activity.

D A Wolf 1, L J Strobl 1, A Pullner 1, D Eick 1
PMCID: PMC307213  PMID: 7567445

Abstract

Transcriptional activation of the c-myc proto-oncogene is mediated by the transition of promoter proximal, paused RNA polymerase II (pol II) into a processive transcription mode. Using a transcription assay which allows the high resolution mapping of transcriptional complexes in intact nuclei, we have characterized the promoter proximal pause positions of pol II. Pol II paused in a nucleosome-free region close to the transcription start site as well as further downstream, between positions +17 and +52. These pause positions were detected in both transcriptionally active and inactive c-myc genes. Pharmacological inhibition of the C-terminal phosphorylation of the large subunit of pol II did not affect the paused transcription complexes, but had an inhibitory effect on transcription of nucleosomal DNA downstream of position +150. The different properties of pol II proximal and distal to the promoter suggest a model in which c-myc transcription is regulated by the activation of promoter bound polymerases.

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Selected References

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

  1. Bengal E., Flores O., Krauskopf A., Reinberg D., Aloni Y. Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II. Mol Cell Biol. 1991 Mar;11(3):1195–1206. doi: 10.1128/mcb.11.3.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  3. Chodosh L. A., Fire A., Samuels M., Sharp P. A. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J Biol Chem. 1989 Feb 5;264(4):2250–2257. [PubMed] [Google Scholar]
  4. Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A. 1978 May;75(5):2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conaway R. C., Conaway J. W. General initiation factors for RNA polymerase II. Annu Rev Biochem. 1993;62:161–190. doi: 10.1146/annurev.bi.62.070193.001113. [DOI] [PubMed] [Google Scholar]
  6. Dahmus M. E. The role of multisite phosphorylation in the regulation of RNA polymerase II activity. Prog Nucleic Acid Res Mol Biol. 1994;48:143–179. doi: 10.1016/s0079-6603(08)60855-7. [DOI] [PubMed] [Google Scholar]
  7. Dubois M. F., Nguyen V. T., Dahmus M. E., Pagès G., Pouysségur J., Bensaude O. Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases. EMBO J. 1994 Oct 17;13(20):4787–4797. doi: 10.1002/j.1460-2075.1994.tb06804.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eick D., Bornkamm G. W. Transcriptional arrest within the first exon is a fast control mechanism in c-myc gene expression. Nucleic Acids Res. 1986 Nov 11;14(21):8331–8346. doi: 10.1093/nar/14.21.8331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eick D., Kohlhuber F., Wolf D. A., Strobl L. J. Activation of pausing RNA polymerases by nuclear run-on experiments. Anal Biochem. 1994 May 1;218(2):347–351. doi: 10.1006/abio.1994.1190. [DOI] [PubMed] [Google Scholar]
  10. Eick D., Wedel A., Heumann H. From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases. Trends Genet. 1994 Aug;10(8):292–296. doi: 10.1016/0168-9525(90)90013-v. [DOI] [PubMed] [Google Scholar]
  11. Feaver W. J., Gileadi O., Li Y., Kornberg R. D. CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell. 1991 Dec 20;67(6):1223–1230. doi: 10.1016/0092-8674(91)90298-d. [DOI] [PubMed] [Google Scholar]
  12. Feaver W. J., Svejstrup J. Q., Henry N. L., Kornberg R. D. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell. 1994 Dec 16;79(6):1103–1109. doi: 10.1016/0092-8674(94)90040-x. [DOI] [PubMed] [Google Scholar]
  13. Flores O., Maldonado E., Reinberg D. Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II. J Biol Chem. 1989 May 25;264(15):8913–8921. [PubMed] [Google Scholar]
  14. Gazin C., Dupont de Dinechin S., Hampe A., Masson J. M., Martin P., Stehelin D., Galibert F. Nucleotide sequence of the human c-myc locus: provocative open reading frame within the first exon. EMBO J. 1984 Feb;3(2):383–387. doi: 10.1002/j.1460-2075.1984.tb01816.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Izban M. G., Luse D. S. The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII. Genes Dev. 1992 Jul;6(7):1342–1356. doi: 10.1101/gad.6.7.1342. [DOI] [PubMed] [Google Scholar]
  16. Izban M. G., Luse D. S. Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. Genes Dev. 1991 Apr;5(4):683–696. doi: 10.1101/gad.5.4.683. [DOI] [PubMed] [Google Scholar]
  17. Kassavetis G. A., Geiduschek E. P. RNA polymerase marching backward. Science. 1993 Feb 12;259(5097):944–945. doi: 10.1126/science.7679800. [DOI] [PubMed] [Google Scholar]
  18. Kephart D. D., Marshall N. F., Price D. H. Stability of Drosophila RNA polymerase II elongation complexes in vitro. Mol Cell Biol. 1992 May;12(5):2067–2077. doi: 10.1128/mcb.12.5.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  20. Kohlhuber F., Strobl L. J., Eick D. Early down-regulation of c-myc in dimethylsulfoxide-induced mouse erythroleukemia (MEL) cells is mediated at the P1/P2 promoters. Oncogene. 1993 Apr;8(4):1099–1102. [PubMed] [Google Scholar]
  21. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  22. Krumm A., Hickey L. B., Groudine M. Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation. Genes Dev. 1995 Mar 1;9(5):559–572. doi: 10.1101/gad.9.5.559. [DOI] [PubMed] [Google Scholar]
  23. Krumm A., Meulia T., Brunvand M., Groudine M. The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. Genes Dev. 1992 Nov;6(11):2201–2213. doi: 10.1101/gad.6.11.2201. [DOI] [PubMed] [Google Scholar]
  24. Laybourn P. J., Dahmus M. E. Transcription-dependent structural changes in the C-terminal domain of mammalian RNA polymerase subunit IIa/o. J Biol Chem. 1989 Apr 25;264(12):6693–6698. [PubMed] [Google Scholar]
  25. Lis J., Wu C. Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell. 1993 Jul 16;74(1):1–4. doi: 10.1016/0092-8674(93)90286-y. [DOI] [PubMed] [Google Scholar]
  26. Lu H., Zawel L., Fisher L., Egly J. M., Reinberg D. Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II. Nature. 1992 Aug 20;358(6388):641–645. doi: 10.1038/358641a0. [DOI] [PubMed] [Google Scholar]
  27. Marciniak R. A., Sharp P. A. HIV-1 Tat protein promotes formation of more-processive elongation complexes. EMBO J. 1991 Dec;10(13):4189–4196. doi: 10.1002/j.1460-2075.1991.tb04997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marshall N. F., Price D. H. Control of formation of two distinct classes of RNA polymerase II elongation complexes. Mol Cell Biol. 1992 May;12(5):2078–2090. doi: 10.1128/mcb.12.5.2078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meulia T., Krumm A., Groudine M. Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis. Mol Cell Biol. 1993 Sep;13(9):5647–5658. doi: 10.1128/mcb.13.9.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mirkovitch J., Darnell J. E., Jr Mapping of RNA polymerase on mammalian genes in cells and nuclei. Mol Biol Cell. 1992 Oct;3(10):1085–1094. doi: 10.1091/mbc.3.10.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Brien T., Hardin S., Greenleaf A., Lis J. T. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature. 1994 Jul 7;370(6484):75–77. doi: 10.1038/370075a0. [DOI] [PubMed] [Google Scholar]
  32. Payne J. M., Laybourn P. J., Dahmus M. E. The transition of RNA polymerase II from initiation to elongation is associated with phosphorylation of the carboxyl-terminal domain of subunit IIa. J Biol Chem. 1989 Nov 25;264(33):19621–19629. [PubMed] [Google Scholar]
  33. Plet A., Eick D., Blanchard J. M. Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns. Oncogene. 1995 Jan 19;10(2):319–328. [PubMed] [Google Scholar]
  34. Polack A., Feederle R., Klobeck G., Hörtnagel K. Regulatory elements in the immunoglobulin kappa locus induce c-myc activation and the promoter shift in Burkitt's lymphoma cells. EMBO J. 1993 Oct;12(10):3913–3920. doi: 10.1002/j.1460-2075.1993.tb06069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reinberg D., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains. J Biol Chem. 1987 Mar 5;262(7):3331–3337. [PubMed] [Google Scholar]
  36. Roberts S., Bentley D. L. Distinct modes of transcription read through or terminate at the c-myc attenuator. EMBO J. 1992 Mar;11(3):1085–1093. doi: 10.1002/j.1460-2075.1992.tb05147.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rougvie A. E., Lis J. T. Postinitiation transcriptional control in Drosophila melanogaster. Mol Cell Biol. 1990 Nov;10(11):6041–6045. doi: 10.1128/mcb.10.11.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rougvie A. E., Lis J. T. The RNA polymerase II molecule at the 5' end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell. 1988 Sep 9;54(6):795–804. doi: 10.1016/s0092-8674(88)91087-2. [DOI] [PubMed] [Google Scholar]
  39. Roy R., Adamczewski J. P., Seroz T., Vermeulen W., Tassan J. P., Schaeffer L., Nigg E. A., Hoeijmakers J. H., Egly J. M. The MO15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor. Cell. 1994 Dec 16;79(6):1093–1101. doi: 10.1016/0092-8674(94)90039-6. [DOI] [PubMed] [Google Scholar]
  40. Serizawa H., Conaway R. C., Conaway J. W. A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7476–7480. doi: 10.1073/pnas.89.16.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Serizawa H., Mäkelä T. P., Conaway J. W., Conaway R. C., Weinberg R. A., Young R. A. Association of Cdk-activating kinase subunits with transcription factor TFIIH. Nature. 1995 Mar 16;374(6519):280–282. doi: 10.1038/374280a0. [DOI] [PubMed] [Google Scholar]
  42. Shiekhattar R., Mermelstein F., Fisher R. P., Drapkin R., Dynlacht B., Wessling H. C., Morgan D. O., Reinberg D. Cdk-activating kinase complex is a component of human transcription factor TFIIH. Nature. 1995 Mar 16;374(6519):283–287. doi: 10.1038/374283a0. [DOI] [PubMed] [Google Scholar]
  43. Sigler P. B. Transcriptional activation. Acid blobs and negative noodles. Nature. 1988 May 19;333(6170):210–212. doi: 10.1038/333210a0. [DOI] [PubMed] [Google Scholar]
  44. Stevens A., Maupin M. K. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits a HeLa protein kinase that phosphorylates an RNA polymerase II-derived peptide. Biochem Biophys Res Commun. 1989 Mar 15;159(2):508–515. doi: 10.1016/0006-291x(89)90022-3. [DOI] [PubMed] [Google Scholar]
  45. Strobl L. J., Eick D. Hold back of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo. EMBO J. 1992 Sep;11(9):3307–3314. doi: 10.1002/j.1460-2075.1992.tb05409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Strobl L. J., Kohlhuber F., Mautner J., Polack A., Eick D. Absence of a paused transcription complex from the c-myc P2 promoter of the translocation chromosome in Burkitt's lymphoma cells: implication for the c-myc P1/P2 promoter shift. Oncogene. 1993 Jun;8(6):1437–1447. [PubMed] [Google Scholar]
  47. Suzuki M. The heptad repeat in the largest subunit of RNA polymerase II binds by intercalating into DNA. Nature. 1990 Apr 5;344(6266):562–565. doi: 10.1038/344562a0. [DOI] [PubMed] [Google Scholar]
  48. Tamm I., Kikuchi T. Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5750–5754. doi: 10.1073/pnas.76.11.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Usheva A., Maldonado E., Goldring A., Lu H., Houbavi C., Reinberg D., Aloni Y. Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein. Cell. 1992 May 29;69(5):871–881. doi: 10.1016/0092-8674(92)90297-p. [DOI] [PubMed] [Google Scholar]
  50. Weeks J. R., Hardin S. E., Shen J., Lee J. M., Greenleaf A. L. Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev. 1993 Dec;7(12A):2329–2344. doi: 10.1101/gad.7.12a.2329. [DOI] [PubMed] [Google Scholar]
  51. Yankulov K., Blau J., Purton T., Roberts S., Bentley D. L. Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell. 1994 Jun 3;77(5):749–759. doi: 10.1016/0092-8674(94)90058-2. [DOI] [PubMed] [Google Scholar]
  52. Young R. A. RNA polymerase II. Annu Rev Biochem. 1991;60:689–715. doi: 10.1146/annurev.bi.60.070191.003353. [DOI] [PubMed] [Google Scholar]
  53. Zawel L., Reinberg D. Initiation of transcription by RNA polymerase II: a multi-step process. Prog Nucleic Acid Res Mol Biol. 1993;44:67–108. doi: 10.1016/s0079-6603(08)60217-2. [DOI] [PubMed] [Google Scholar]

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