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. 1991 Nov;11(11):5398–5409. doi: 10.1128/mcb.11.11.5398

A heat-labile factor promotes premature 3' end formation in exon 1 of the murine adenosine deaminase gene in a cell-free transcription system.

J W Innis 1, R E Kellems 1
PMCID: PMC361673  PMID: 1717827

Abstract

An elongation block to RNA polymerase II transcription in exon 1 is a major regulatory step in expression of the murine adenosine deaminase (ADA) gene. Previous work in the laboratory identified abundant short transcripts with 3' termini in exon 1 in steady-state RNA from injected oocytes. Using a cell-free system to investigate the mechanism of premature 3' end formation, we found that polymerase II generates prominent ADA transcripts approximately 96 to 100 nucleotides in length which are similar to the major short transcripts found in steady-state RNA from oocytes injected with ADA templates. We have determined that these transcripts are the processed products of 108- to 112-nucleotide precursors. Precursor formation is (i) favored in reactions using circular templates, (ii) not the result of a posttranscriptional processing event, (iii) sensitive to low concentrations of Sarkosyl, and (iv) dependent on a factor(s) which is inactivated in crude extracts at 47 degrees C for 15 min. The cell-free system will allow further characterization of the template and factor requirements involved in the control of premature 3' end formation by RNA polymerase II.

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Selected References

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

  1. Ben-Asher E., Aloni Y. Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation. J Virol. 1984 Oct;52(1):266–276. doi: 10.1128/jvi.52.1.266-276.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender T. P., Thompson C. B., Kuehl W. M. Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation. Science. 1987 Sep 18;237(4821):1473–1476. doi: 10.1126/science.3498214. [DOI] [PubMed] [Google Scholar]
  3. Bentley D. L., Groudine M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature. 1986 Jun 12;321(6071):702–706. doi: 10.1038/321702a0. [DOI] [PubMed] [Google Scholar]
  4. Bentley D. L., Groudine M. Sequence requirements for premature termination of transcription in the human c-myc gene. Cell. 1988 Apr 22;53(2):245–256. doi: 10.1016/0092-8674(88)90386-8. [DOI] [PubMed] [Google Scholar]
  5. Chen Z., Harless M. L., Wright D. A., Kellems R. E. Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene. Mol Cell Biol. 1990 Sep;10(9):4555–4564. doi: 10.1128/mcb.10.9.4555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chinsky J. M., Maa M. C., Ramamurthy V., Kellems R. E. Adenosine deaminase gene expression. Tissue-dependent regulation of transcriptional elongation. J Biol Chem. 1989 Aug 25;264(24):14561–14565. [PubMed] [Google Scholar]
  7. Cullen B. R. The HIV-1 Tat protein: an RNA sequence-specific processivity factor? Cell. 1990 Nov 16;63(4):655–657. doi: 10.1016/0092-8674(90)90129-3. [DOI] [PubMed] [Google Scholar]
  8. Culotta V. C., Wides R. J., Sollner-Webb B. Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors. Mol Cell Biol. 1985 Jul;5(7):1582–1590. doi: 10.1128/mcb.5.7.1582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fort P., Rech J., Vie A., Piechaczyk M., Bonnieu A., Jeanteur P., Blanchard J. M. Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation. Nucleic Acids Res. 1987 Jul 24;15(14):5657–5667. doi: 10.1093/nar/15.14.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grayhack E. J., Roberts J. W. The phage lambda Q gene product: activity of a transcription antiterminator in vitro. Cell. 1982 Sep;30(2):637–648. doi: 10.1016/0092-8674(82)90260-4. [DOI] [PubMed] [Google Scholar]
  12. Grayhack E. J., Yang X. J., Lau L. F., Roberts J. W. Phage lambda gene Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site. Cell. 1985 Aug;42(1):259–269. doi: 10.1016/s0092-8674(85)80121-5. [DOI] [PubMed] [Google Scholar]
  13. Haley J. D., Waterfield M. D. Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis. J Biol Chem. 1991 Jan 25;266(3):1746–1753. [PubMed] [Google Scholar]
  14. Hawley D. K., Roeder R. G. Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II. J Biol Chem. 1985 Jul 5;260(13):8163–8172. [PubMed] [Google Scholar]
  15. Hay N., Aloni Y. Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B. Nucleic Acids Res. 1984 Feb 10;12(3):1401–1414. doi: 10.1093/nar/12.3.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hay N., Skolnik-David H., Aloni Y. Attenuation in the control of SV40 gene expression. Cell. 1982 May;29(1):183–193. doi: 10.1016/0092-8674(82)90102-7. [DOI] [PubMed] [Google Scholar]
  17. Kao S. Y., Calman A. F., Luciw P. A., Peterlin B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987 Dec 3;330(6147):489–493. doi: 10.1038/330489a0. [DOI] [PubMed] [Google Scholar]
  18. Krystal G., Birrer M., Way J., Nau M., Sausville E., Thompson C., Minna J., Battey J. Multiple mechanisms for transcriptional regulation of the myc gene family in small-cell lung cancer. Mol Cell Biol. 1988 Aug;8(8):3373–3381. doi: 10.1128/mcb.8.8.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuhn A., Grummt I. 3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction. Genes Dev. 1989 Feb;3(2):224–231. doi: 10.1101/gad.3.2.224. [DOI] [PubMed] [Google Scholar]
  20. Lamb N. J., Fernandez A., Tourkine N., Jeanteur P., Blanchard J. M. Demonstration in living cells of an intragenic negative regulatory element within the rodent c-fos gene. Cell. 1990 May 4;61(3):485–496. doi: 10.1016/0092-8674(90)90530-r. [DOI] [PubMed] [Google Scholar]
  21. Laspia M. F., Rice A. P., Mathews M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell. 1989 Oct 20;59(2):283–292. doi: 10.1016/0092-8674(89)90290-0. [DOI] [PubMed] [Google Scholar]
  22. Laspia M. F., Rice A. P., Mathews M. B. Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation. Genes Dev. 1990 Dec;4(12B):2397–2408. doi: 10.1101/gad.4.12b.2397. [DOI] [PubMed] [Google Scholar]
  23. Lazinski D., Grzadzielska E., Das A. Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell. 1989 Oct 6;59(1):207–218. doi: 10.1016/0092-8674(89)90882-9. [DOI] [PubMed] [Google Scholar]
  24. Maa M. C., Chinsky J. M., Ramamurthy V., Martin B. D., Kellems R. E. Identification of transcription stop sites at the 5' and 3' ends of the murine adenosine deaminase gene. J Biol Chem. 1990 Jul 25;265(21):12513–12519. [PubMed] [Google Scholar]
  25. Maderious A., Chen-Kiang S. Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5931–5935. doi: 10.1073/pnas.81.19.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marciniak R. A., Calnan B. J., Frankel A. D., Sharp P. A. HIV-1 Tat protein trans-activates transcription in vitro. Cell. 1990 Nov 16;63(4):791–802. doi: 10.1016/0092-8674(90)90145-5. [DOI] [PubMed] [Google Scholar]
  27. Marciniak R. A., Garcia-Blanco M. A., Sharp P. A. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1990 May;87(9):3624–3628. doi: 10.1073/pnas.87.9.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McGeady M. L., Wood T. G., Maizel J. V., Vande Woude G. F. Sequences upstream from the mouse c-mos oncogene may function as a transcription termination signal. DNA. 1986 Aug;5(4):289–298. doi: 10.1089/dna.1986.5.289. [DOI] [PubMed] [Google Scholar]
  29. Mizutani M., Ohta T., Watanabe H., Handa H., Hirose S. Negative supercoiling of DNA facilitates an interaction between transcription factor IID and the fibroin gene promoter. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):718–722. doi: 10.1073/pnas.88.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mott J. E., Galloway J. L., Platt T. Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination. EMBO J. 1985 Jul;4(7):1887–1891. doi: 10.1002/j.1460-2075.1985.tb03865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakajima N., Horikoshi M., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol Cell Biol. 1988 Oct;8(10):4028–4040. doi: 10.1128/mcb.8.10.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  33. Ramamurthy V., Maa M. C., Harless M. L., Wright D. A., Kellems R. E. Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene. Mol Cell Biol. 1990 Apr;10(4):1484–1491. doi: 10.1128/mcb.10.4.1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Resnekov O., Aloni Y. RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro. Proc Natl Acad Sci U S A. 1989 Jan;86(1):12–16. doi: 10.1073/pnas.86.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Richardson J. P. Preventing the synthesis of unused transcripts by Rho factor. Cell. 1991 Mar 22;64(6):1047–1049. doi: 10.1016/0092-8674(91)90257-y. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Sancar G. B., Sancar A., Little J. W., Rupp W. D. The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters. Cell. 1982 Mar;28(3):523–530. doi: 10.1016/0092-8674(82)90207-0. [DOI] [PubMed] [Google Scholar]
  39. Schneider-Schaulies J., Schimpl A., Wecker E. Kinetics of cellular oncogene expression in mouse lymphocytes. II. Regulation of c-fos and c-myc gene expression. Eur J Immunol. 1987 May;17(5):713–718. doi: 10.1002/eji.1830170521. [DOI] [PubMed] [Google Scholar]
  40. Sharp P. A., Marciniak R. A. HIV TAR: an RNA enhancer? Cell. 1989 Oct 20;59(2):229–230. doi: 10.1016/0092-8674(89)90279-1. [DOI] [PubMed] [Google Scholar]
  41. Shen C. C., Shen C. K. Specificity and flexibility of the recognition of DNA helical structure by eukaryotic topoisomerase I. J Mol Biol. 1990 Mar 5;212(1):67–78. doi: 10.1016/0022-2836(90)90305-6. [DOI] [PubMed] [Google Scholar]
  42. SivaRaman L., Reines D., Kane C. M. Purified elongation factor SII is sufficient to promote read-through by purified RNA polymerase II at specific termination sites in the human histone H3.3 gene. J Biol Chem. 1990 Aug 25;265(24):14554–14560. [PubMed] [Google Scholar]
  43. Skarnes W. C., Tessier D. C., Acheson N. H. RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA. J Mol Biol. 1988 Sep 5;203(1):153–171. doi: 10.1016/0022-2836(88)90099-x. [DOI] [PubMed] [Google Scholar]
  44. Spencer C. A., Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990 Jun;5(6):777–785. [PubMed] [Google Scholar]
  45. Spitzner J. R., Chung I. K., Muller M. T. Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats. Nucleic Acids Res. 1990 Jan 11;18(1):1–11. doi: 10.1093/nar/18.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Toohey M. G., Jones K. A. In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions. Genes Dev. 1989 Mar;3(3):265–282. doi: 10.1101/gad.3.3.265. [DOI] [PubMed] [Google Scholar]
  47. Watson R. J. A transcriptional arrest mechanism involved in controlling constitutive levels of mouse c-myb mRNA. Oncogene. 1988 Mar;2(3):267–272. [PubMed] [Google Scholar]
  48. Weeks K. M., Ampe C., Schultz S. C., Steitz T. A., Crothers D. M. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. doi: 10.1126/science.2205002. [DOI] [PubMed] [Google Scholar]
  49. Wiest D. K., Hawley D. K. In vitro analysis of a transcription termination site for RNA polymerase II. Mol Cell Biol. 1990 Nov;10(11):5782–5795. doi: 10.1128/mcb.10.11.5782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yanofsky C. Transcription attenuation. J Biol Chem. 1988 Jan 15;263(2):609–612. [PubMed] [Google Scholar]

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