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. 1992 May;12(5):2250–2259. doi: 10.1128/mcb.12.5.2250

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

A B Buermeyer 1, N E Thompson 1, L A Strasheim 1, R R Burgess 1, P J Farnham 1
PMCID: PMC364397  PMID: 1569952

Abstract

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

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Selected References

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

  1. Allison L. A., Ingles C. J. Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2794–2798. doi: 10.1073/pnas.86.8.2794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison L. A., Wong J. K., Fitzpatrick V. D., Moyle M., Ingles C. J. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol Cell Biol. 1988 Jan;8(1):321–329. doi: 10.1128/mcb.8.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arias J. A., Peterson S. R., Dynan W. S. Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation. J Biol Chem. 1991 May 5;266(13):8055–8061. [PubMed] [Google Scholar]
  4. Bartolomei M. S., Halden N. F., Cullen C. R., Corden J. L. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II. Mol Cell Biol. 1988 Jan;8(1):330–339. doi: 10.1128/mcb.8.1.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beck T. W., Brennscheidt U., Sithanandam G., Cleveland J., Rapp U. R. Molecular organization of the human Raf-1 promoter region. Mol Cell Biol. 1990 Jul;10(7):3325–3333. doi: 10.1128/mcb.10.7.3325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borrelli M. J., Mackey M. A., Dewey W. C. A method for freezing synchronous mitotic and G1 cells. Exp Cell Res. 1987 Jun;170(2):363–368. doi: 10.1016/0014-4827(87)90313-2. [DOI] [PubMed] [Google Scholar]
  7. Buratowski S., Sharp P. A. Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit. Mol Cell Biol. 1990 Oct;10(10):5562–5564. doi: 10.1128/mcb.10.10.5562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cisek L. J., Corden J. L. Phosphorylation of RNA polymerase by the murine homologue of the cell-cycle control protein cdc2. Nature. 1989 Jun 29;339(6227):679–684. doi: 10.1038/339679a0. [DOI] [PubMed] [Google Scholar]
  9. Corden J. L., Cadena D. L., Ahearn J. M., Jr, Dahmus M. E. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7934–7938. doi: 10.1073/pnas.82.23.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Corden J. L. Tails of RNA polymerase II. Trends Biochem Sci. 1990 Oct;15(10):383–387. doi: 10.1016/0968-0004(90)90236-5. [DOI] [PubMed] [Google Scholar]
  11. Dahmus M. E., Kedinger C. Transcription of adenovirus-2 major late promoter inhibited by monoclonal antibody directed against RNA polymerases IIO and IIA. J Biol Chem. 1983 Feb 25;258(4):2303–2307. [PubMed] [Google Scholar]
  12. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farnham P. J., Abrams J. M., Schimke R. T. Opposite-strand RNAs from the 5' flanking region of the mouse dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3978–3982. doi: 10.1073/pnas.82.12.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Farnham P. J., Cornwell M. M. Sp1 activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component. Gene Expr. 1991 May;1(2):137–148. [PMC free article] [PubMed] [Google Scholar]
  15. Farnham P. J., Kollmar R. Characterization of the 5' end of the growth-regulated Syrian hamster CAD gene. Cell Growth Differ. 1990 Apr;1(4):179–189. [PubMed] [Google Scholar]
  16. Farnham P. J., Means A. L. Sequences downstream of the transcription initiation site modulate the activity of the murine dihydrofolate reductase promoter. Mol Cell Biol. 1990 Apr;10(4):1390–1398. doi: 10.1128/mcb.10.4.1390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Farnham P. J., Schimke R. T. In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene. Mol Cell Biol. 1986 Jul;6(7):2392–2401. doi: 10.1128/mcb.6.7.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hodo H. G., 3rd, Blatti S. P. Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II. Biochemistry. 1977 May 31;16(11):2334–2343. doi: 10.1021/bi00630a005. [DOI] [PubMed] [Google Scholar]
  20. Kim W. Y., Dahmus M. E. Purification of RNA polymerase IIO from calf thymus. J Biol Chem. 1988 Dec 15;263(35):18880–18885. [PubMed] [Google Scholar]
  21. Kim W. Y., Dahmus M. E. The major late promoter of adenovirus-2 is accurately transcribed by RNA polymerases IIO, IIA, and IIB. J Biol Chem. 1989 Feb 25;264(6):3169–3176. [PubMed] [Google Scholar]
  22. Kruger W., Herskowitz I. A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMG1. Mol Cell Biol. 1991 Aug;11(8):4135–4146. doi: 10.1128/mcb.11.8.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laybourn P. J., Dahmus M. E. Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription. J Biol Chem. 1990 Aug 5;265(22):13165–13173. [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. Lee J. M., Greenleaf A. L. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II. Proc Natl Acad Sci U S A. 1989 May;86(10):3624–3628. doi: 10.1073/pnas.86.10.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee J. M., Greenleaf A. L. CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. Gene Expr. 1991 May;1(2):149–167. [PMC free article] [PubMed] [Google Scholar]
  27. Liao S. M., Taylor I. C., Kingston R. E., Young R. A. RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro. Genes Dev. 1991 Dec;5(12B):2431–2440. doi: 10.1101/gad.5.12b.2431. [DOI] [PubMed] [Google Scholar]
  28. Linton J. P., Yen J. Y., Selby E., Chen Z., Chinsky J. M., Liu K., Kellems R. E., Crouse G. F. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene. Mol Cell Biol. 1989 Jul;9(7):3058–3072. doi: 10.1128/mcb.9.7.3058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Means A. L., Farnham P. J. Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site. Mol Cell Biol. 1990 Feb;10(2):653–661. doi: 10.1128/mcb.10.2.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Means A. L., Slansky J. E., McMahon S. L., Knuth M. W., Farnham P. J. The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter. Mol Cell Biol. 1992 Mar;12(3):1054–1063. doi: 10.1128/mcb.12.3.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miyamoto M., Fujita T., Kimura Y., Maruyama M., Harada H., Sudo Y., Miyata T., Taniguchi T. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell. 1988 Sep 9;54(6):903–913. doi: 10.1016/s0092-8674(88)91307-4. [DOI] [PubMed] [Google Scholar]
  32. Nonet M., Sweetser D., Young R. A. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987 Sep 11;50(6):909–915. doi: 10.1016/0092-8674(87)90517-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Peterson C. L., Kruger W., Herskowitz I. A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1. Cell. 1991 Mar 22;64(6):1135–1143. doi: 10.1016/0092-8674(91)90268-4. [DOI] [PubMed] [Google Scholar]
  35. Sawadogo M., Sentenac A. RNA polymerase B (II) and general transcription factors. Annu Rev Biochem. 1990;59:711–754. doi: 10.1146/annurev.bi.59.070190.003431. [DOI] [PubMed] [Google Scholar]
  36. Scafe C., Chao D., Lopes J., Hirsch J. P., Henry S., Young R. A. RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals. Nature. 1990 Oct 4;347(6292):491–494. doi: 10.1038/347491a0. [DOI] [PubMed] [Google Scholar]
  37. Schilling L. J., Farnham P. J. Identification of a new promoter upstream of the murine dihydrofolate reductase gene. Mol Cell Biol. 1989 Oct;9(10):4568–4570. doi: 10.1128/mcb.9.10.4568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Steinberg T. H., Mathews D. E., Durbin R. D., Burgess R. R. Tagetitoxin: a new inhibitor of eukaryotic transcription by RNA polymerase III. J Biol Chem. 1990 Jan 5;265(1):499–505. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Thompson N. E., Aronson D. B., Burgess R. R. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody. J Biol Chem. 1990 Apr 25;265(12):7069–7077. [PubMed] [Google Scholar]
  42. Thompson N. E., Steinberg T. H., Aronson D. B., Burgess R. R. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem. 1989 Jul 5;264(19):11511–11520. [PubMed] [Google Scholar]
  43. 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]
  44. Zandomeni R., Zandomeni M. C., Shugar D., Weinmann R. Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription. J Biol Chem. 1986 Mar 5;261(7):3414–3419. [PubMed] [Google Scholar]
  45. Zehring W. A., Greenleaf A. L. The carboxyl-terminal repeat domain of RNA polymerase II is not required for transcription factor Sp1 to function in vitro. J Biol Chem. 1990 May 25;265(15):8351–8353. [PubMed] [Google Scholar]
  46. Zehring W. A., Lee J. M., Weeks J. R., Jokerst R. S., Greenleaf A. L. The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3698–3702. doi: 10.1073/pnas.85.11.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]

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