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. 1993 Sep;13(9):5918–5927. doi: 10.1128/mcb.13.9.5918

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

Z Zamrod 1, C M Tyree 1, Y Song 1, W E Stumph 1
PMCID: PMC360340  PMID: 8355718

Abstract

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

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Selected References

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

  1. Beck E., Jorcano J. L., Alonso A. Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences. J Mol Biol. 1984 Mar 15;173(4):539–542. doi: 10.1016/0022-2836(84)90397-8. [DOI] [PubMed] [Google Scholar]
  2. Bungert J., Kober I., Düring F., Seifart K. H. Transcription factor eUSF is an essential component of isolated transcription complexes on the duck histone H5 gene and it mediates the interaction of TFIID with a TATA-deficient promoter. J Mol Biol. 1992 Feb 20;223(4):885–898. doi: 10.1016/0022-2836(92)90250-n. [DOI] [PubMed] [Google Scholar]
  3. Carcamo J., Buckbinder L., Reinberg D. The initiator directs the assembly of a transcription factor IID-dependent transcription complex. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8052–8056. doi: 10.1073/pnas.88.18.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu B. C., Orgel L. E. Binding of hairpin and dumbbell DNA to transcription factors. Nucleic Acids Res. 1991 Dec 25;19(24):6958–6958. doi: 10.1093/nar/19.24.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Comai L., Tanese N., Tjian R. The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell. 1992 Mar 6;68(5):965–976. doi: 10.1016/0092-8674(92)90039-f. [DOI] [PubMed] [Google Scholar]
  6. Cormack B. P., Struhl K. The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. Cell. 1992 May 15;69(4):685–696. doi: 10.1016/0092-8674(92)90232-2. [DOI] [PubMed] [Google Scholar]
  7. Dahlberg J. E., Lund E. How does III x II make U6? Science. 1991 Dec 6;254(5037):1462–1463. doi: 10.1126/science.1962205. [DOI] [PubMed] [Google Scholar]
  8. Das G., Henning D., Reddy R. Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes. J Biol Chem. 1987 Jan 25;262(3):1187–1193. [PubMed] [Google Scholar]
  9. Dynlacht B. D., Hoey T., Tjian R. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell. 1991 Aug 9;66(3):563–576. doi: 10.1016/0092-8674(81)90019-2. [DOI] [PubMed] [Google Scholar]
  10. Gunderson S. I., Knuth M. W., Burgess R. R. The human U1 snRNA promoter correctly initiates transcription in vitro and is activated by PSE1. Genes Dev. 1990 Dec;4(12A):2048–2060. doi: 10.1101/gad.4.12a.2048. [DOI] [PubMed] [Google Scholar]
  11. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  12. Heiermann R., Pongs O. In vitro transcription with extracts of nuclei of Drosophila embryos. Nucleic Acids Res. 1985 Apr 25;13(8):2709–2730. doi: 10.1093/nar/13.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hernandez N., Lucito R. Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J. 1988 Oct;7(10):3125–3134. doi: 10.1002/j.1460-2075.1988.tb03179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hernandez N., Weiner A. M. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell. 1986 Oct 24;47(2):249–258. doi: 10.1016/0092-8674(86)90447-2. [DOI] [PubMed] [Google Scholar]
  15. Hoey T., Dynlacht B. D., Peterson M. G., Pugh B. F., Tjian R. Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID. Cell. 1990 Jun 29;61(7):1179–1186. doi: 10.1016/0092-8674(90)90682-5. [DOI] [PubMed] [Google Scholar]
  16. Hoffman A., Sinn E., Yamamoto T., Wang J., Roy A., Horikoshi M., Roeder R. G. Highly conserved core domain and unique N terminus with presumptive regulatory motifs in a human TATA factor (TFIID). Nature. 1990 Jul 26;346(6282):387–390. doi: 10.1038/346387a0. [DOI] [PubMed] [Google Scholar]
  17. Kadonaga J. T. Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription. J Biol Chem. 1990 Feb 15;265(5):2624–2631. [PubMed] [Google Scholar]
  18. Kamakaka R. T., Tyree C. M., Kadonaga J. T. Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1024–1028. doi: 10.1073/pnas.88.3.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kassavetis G. A., Joazeiro C. A., Pisano M., Geiduschek E. P., Colbert T., Hahn S., Blanco J. A. The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. Cell. 1992 Dec 11;71(6):1055–1064. doi: 10.1016/0092-8674(92)90399-w. [DOI] [PubMed] [Google Scholar]
  20. Kejzlarová-Lepesant J., Brock H. W., Moreau J., Dubertret M. L., Billault A., Lepesant J. A. A complete and a truncated U1 snRNA gene of Drosophila melanogaster are found as inverted repeats at region 82E of the polytene chromosomes. Nucleic Acids Res. 1984 Dec 11;12(23):8835–8846. doi: 10.1093/nar/12.23.8835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lescure A., Carbon P., Krol A. The different positioning of the proximal sequence element in the Xenopus RNA polymerase II and III snRNA promoters is a key determinant which confers RNA polymerase III specificity. Nucleic Acids Res. 1991 Feb 11;19(3):435–441. doi: 10.1093/nar/19.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lo P. C., Mount S. M. Drosophila melanogaster genes for U1 snRNA variants and their expression during development. Nucleic Acids Res. 1990 Dec 11;18(23):6971–6979. doi: 10.1093/nar/18.23.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lobo S. M., Hernandez N. A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell. 1989 Jul 14;58(1):55–67. doi: 10.1016/0092-8674(89)90402-9. [DOI] [PubMed] [Google Scholar]
  24. Lobo S. M., Ifill S., Hernandez N. cis-acting elements required for RNA polymerase II and III transcription in the human U2 and U6 snRNA promoters. Nucleic Acids Res. 1990 May 25;18(10):2891–2899. doi: 10.1093/nar/18.10.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lobo S. M., Lister J., Sullivan M. L., Hernandez N. The cloned RNA polymerase II transcription factor IID selects RNA polymerase III to transcribe the human U6 gene in vitro. Genes Dev. 1991 Aug;5(8):1477–1489. doi: 10.1101/gad.5.8.1477. [DOI] [PubMed] [Google Scholar]
  26. Lobo S. M., Tanaka M., Sullivan M. L., Hernandez N. A TBP complex essential for transcription from TATA-less but not TATA-containing RNA polymerase III promoters is part of the TFIIIB fraction. Cell. 1992 Dec 11;71(6):1029–1040. doi: 10.1016/0092-8674(92)90397-u. [DOI] [PubMed] [Google Scholar]
  27. Lund E., Dahlberg J. E. In vitro synthesis of vertebrate U1 snRNA. EMBO J. 1989 Jan;8(1):287–292. doi: 10.1002/j.1460-2075.1989.tb03375.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mattaj I. W., Dathan N. A., Parry H. D., Carbon P., Krol A. Changing the RNA polymerase specificity of U snRNA gene promoters. Cell. 1988 Nov 4;55(3):435–442. doi: 10.1016/0092-8674(88)90029-3. [DOI] [PubMed] [Google Scholar]
  29. Morris G. F., Price D. H., Marzluff W. F. Synthesis of U1 RNA in a DNA-dependent system from sea urchin embryos. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3674–3678. doi: 10.1073/pnas.83.11.3674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murphy J. T., Skuzeski J. T., Lund E., Steinberg T. H., Burgess R. R., Dahlberg J. E. Functional elements of the human U1 RNA promoter. Identification of five separate regions required for efficient transcription and template competition. J Biol Chem. 1987 Feb 5;262(4):1795–1803. [PubMed] [Google Scholar]
  31. Neuman de Vegvar H. E., Dahlberg J. E. Initiation and termination of human U1 RNA transcription requires the concerted action of multiple flanking elements. Nucleic Acids Res. 1989 Nov 25;17(22):9305–9318. doi: 10.1093/nar/17.22.9305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pugh B. F., Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev. 1991 Nov;5(11):1935–1945. doi: 10.1101/gad.5.11.1935. [DOI] [PubMed] [Google Scholar]
  33. Reddy R. Transcription of a U6 small nuclear RNA gene in vitro. Transcription of a mouse U6 small nuclear RNA gene in vitro by RNA polymerase III is dependent on transcription factor(s) different from transcription factors IIIA, IIIB, and IIIC. J Biol Chem. 1988 Nov 5;263(31):15980–15984. [PubMed] [Google Scholar]
  34. Rigby P. W. Three in one and one in three: it all depends on TBP. Cell. 1993 Jan 15;72(1):7–10. doi: 10.1016/0092-8674(93)90042-o. [DOI] [PubMed] [Google Scholar]
  35. Saba J. A., Busch H., Wright D., Reddy R. Isolation and characterization of two putative full-length Drosophila U4 small nuclear RNA genes. J Biol Chem. 1986 Jul 5;261(19):8750–8753. [PubMed] [Google Scholar]
  36. Schultz M. C., Reeder R. H., Hahn S. Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters. Cell. 1992 May 15;69(4):697–702. doi: 10.1016/0092-8674(92)90233-3. [DOI] [PubMed] [Google Scholar]
  37. Simmen K. A., Bernués J., Lewis J. D., Mattaj I. W. Cofractionation of the TATA-binding protein with the RNA polymerase III transcription factor TFIIIB. Nucleic Acids Res. 1992 Nov 25;20(22):5889–5898. doi: 10.1093/nar/20.22.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simmen K. A., Bernués J., Parry H. D., Stunnenberg H. G., Berkenstam A., Cavallini B., Egly J. M., Mattaj I. W. TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III. EMBO J. 1991 Jul;10(7):1853–1862. doi: 10.1002/j.1460-2075.1991.tb07711.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Simmen K. A., Waldschmidt R., Bernués J., Parry H. D., Seifart K. H., Mattaj I. W. Proximal sequence element factor binding and species specificity in vertebrate U6 snRNA promoters. J Mol Biol. 1992 Feb 20;223(4):873–884. doi: 10.1016/0022-2836(92)90249-j. [DOI] [PubMed] [Google Scholar]
  40. Sluder A. E., Price D. H., Greenleaf A. L. An activity necessary for in vitro transcription is a DNase inhibitor. Biochimie. 1987 Nov-Dec;69(11-12):1199–1205. doi: 10.1016/0300-9084(87)90147-7. [DOI] [PubMed] [Google Scholar]
  41. Soeller W. C., Poole S. J., Kornberg T. In vitro transcription of the Drosophila engrailed gene. Genes Dev. 1988 Jan;2(1):68–81. doi: 10.1101/gad.2.1.68. [DOI] [PubMed] [Google Scholar]
  42. Southgate C., Busslinger M. In vivo and in vitro expression of U7 snRNA genes: cis- and trans-acting elements required for RNA polymerase II-directed transcription. EMBO J. 1989 Feb;8(2):539–549. doi: 10.1002/j.1460-2075.1989.tb03408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stefanovic B., Marzluff W. F. Characterization of two developmentally regulated sea urchin U2 small nuclear RNA promoters: a common required TATA sequence and independent proximal and distal elements. Mol Cell Biol. 1992 Feb;12(2):650–660. doi: 10.1128/mcb.12.2.650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Taggart A. K., Fisher T. S., Pugh B. F. The TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB. Cell. 1992 Dec 11;71(6):1015–1028. doi: 10.1016/0092-8674(92)90396-t. [DOI] [PubMed] [Google Scholar]
  46. Waibel F., Filipowicz W. RNA-polymerase specificity of transcription of Arabidopsis U snRNA genes determined by promoter element spacing. Nature. 1990 Jul 12;346(6280):199–202. doi: 10.1038/346199a0. [DOI] [PubMed] [Google Scholar]
  47. Waldschmidt R., Wanandi I., Seifart K. H. Identification of transcription factors required for the expression of mammalian U6 genes in vitro. EMBO J. 1991 Sep;10(9):2595–2603. doi: 10.1002/j.1460-2075.1991.tb07801.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wampler S. L., Tyree C. M., Kadonaga J. T. Fractionation of the general RNA polymerase II transcription factors from Drosophila embryos. J Biol Chem. 1990 Dec 5;265(34):21223–21231. [PubMed] [Google Scholar]
  49. White R. J., Jackson S. P. Mechanism of TATA-binding protein recruitment to a TATA-less class III promoter. Cell. 1992 Dec 11;71(6):1041–1053. doi: 10.1016/0092-8674(92)90398-v. [DOI] [PubMed] [Google Scholar]
  50. White R. J., Jackson S. P., Rigby P. W. A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1949–1953. doi: 10.1073/pnas.89.5.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou Q., Lieberman P. M., Boyer T. G., Berk A. J. Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter. Genes Dev. 1992 Oct;6(10):1964–1974. doi: 10.1101/gad.6.10.1964. [DOI] [PubMed] [Google Scholar]
  52. de Vegvar H. E., Lund E., Dahlberg J. E. 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. Cell. 1986 Oct 24;47(2):259–266. doi: 10.1016/0092-8674(86)90448-4. [DOI] [PubMed] [Google Scholar]

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