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PMID: 1992453 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 88 ·No. 3 ·1991-02-01 ·Pages 1024-8

Kamakaka RT, Tyree CM, Kadonaga JT

Abstract

We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Krüppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of approximately 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired.

MeSH Terms
Animals Cell Nucleus/metabolism DNA-Binding Proteins/metabolism Drosophila/embryology,genetics Embryo, Nonmammalian/metabolism Genes Kinetics Promoter Regions, Genetic RNA Polymerase II/metabolism RNA, Small Nuclear/genetics Transcription, Genetic
Chemicals
DNA-Binding Proteins RNA, Small Nuclear RNA Polymerase II
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kamakaka R T
Department of Biology, University of California, San Diego, La Jolla 92093.
Tyree C M
Kadonaga J T
References (40)
40 references, click to expand
  1. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  2. How eukaryotic transcriptional activators work.
    Nature. 1988 Oct 20;335(6192):683-9 PMID: 3050531
  3. 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):469-84 PMID: 498279
  4. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.
    Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855-9 PMID: 6933441
  5. Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing.
    Nucleic Acids Res. 1981 Dec 11;9(23):6351-68 PMID: 6172778
  6. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
    Nucleic Acids Res. 1983 Mar 11;11(5):1475-89 PMID: 6828386
  7. A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity.
    Cell. 1984 Feb;36(2):357-69 PMID: 6537904
  8. The messenger RNA for alcohol dehydrogenase in Drosophila melanogaster differs in its 5' end in different developmental stages.
    Cell. 1983 May;33(1):125-33 PMID: 6432335
  9. Molecular genetics of Krüppel, a gene required for segmentation of the Drosophila embryo.
    Nature. 1985 Jan 3-9;313(5997):27-32 PMID: 3917552
  10. In vitro transcription with extracts of nuclei of Drosophila embryos.
    Nucleic Acids Res. 1985 Apr 25;13(8):2709-30 PMID: 2987864
  11. Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II.
    J Biol Chem. 1985 Jul 5;260(13):8163-72 PMID: 2409080
  12. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay.
    Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394-8 PMID: 3925456
  13. Synthesis of U1 RNA in a DNA-dependent system from sea urchin embryos.
    Proc Natl Acad Sci U S A. 1986 Jun;83(11):3674-8 PMID: 3459149
  14. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1.
    Science. 1986 Oct 3;234(4772):47-52 PMID: 3529394
  15. Tissue-specific in vitro transcription from the mouse albumin promoter.
    Cell. 1986 Dec 5;47(5):767-76 PMID: 3779841
  16. Functional steps in transcription initiation and reinitiation from the major late promoter in a HeLa nuclear extract.
    J Biol Chem. 1987 Mar 15;262(8):3452-61 PMID: 2434502
  17. Promoter specificity and modulation of RNA polymerase II transcription.
    FASEB J. 1989 Apr;3(6):1723-33 PMID: 2649403
  18. Novel Jun- and Fos-related proteins in Drosophila are functionally homologous to enhancer factor AP-1.
    EMBO J. 1988 Dec 20;7(13):4265-73 PMID: 3149584
  19. In vitro synthesis of vertebrate U1 snRNA.
    EMBO J. 1989 Jan;8(1):287-92 PMID: 2714253
  20. 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):539-49 PMID: 2721491
  21. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins.
    Science. 1989 Jul 28;245(4916):371-8 PMID: 2667136
  22. Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.
    Mol Cell Biol. 1989 Nov;9(11):4746-9 PMID: 2557540
  23. Transcription and cap trimethylation of a nematode spliced leader RNA in a cell-free system.
    Proc Natl Acad Sci U S A. 1990 Jan;87(2):709-13 PMID: 2300557
  24. A potent GAL4 derivative activates transcription at a distance in vitro.
    Science. 1990 Feb 9;247(4943):710-2 PMID: 2405489
  25. Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription.
    J Biol Chem. 1990 Feb 15;265(5):2624-31 PMID: 1689291
  26. A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives.
    Nature. 1990 May 24;345(6273):361-4 PMID: 2160609
  27. Functional domains and upstream activation properties of cloned human TATA binding protein.
    Science. 1990 Jun 29;248(4963):1625-30 PMID: 2363050
  28. Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID.
    Cell. 1990 Jun 29;61(7):1179-86 PMID: 2194666
  29. Mechanism of transcriptional activation by Sp1: evidence for coactivators.
    Cell. 1990 Jun 29;61(7):1187-97 PMID: 2194667
  30. Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors.
    Cell. 1990 Jun 29;61(7):1199-208 PMID: 2163758
  31. A novel mediator between activator proteins and the RNA polymerase II transcription apparatus.
    Cell. 1990 Jun 29;61(7):1209-15 PMID: 2163759
  32. Fos and jun cooperate in transcriptional regulation via heterologous activation domains.
    Mol Cell Biol. 1990 Oct;10(10):5532-5 PMID: 2119000
  33. Fractionation of the general RNA polymerase II transcription factors from Drosophila embryos.
    J Biol Chem. 1990 Dec 5;265(34):21223-31 PMID: 2250021
  34. Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae.
    Proc Natl Acad Sci U S A. 1987 Dec;84(24):8839-43 PMID: 3321057
  35. A high-efficiency HeLa cell nuclear transcription extract.
    DNA. 1988 Jan-Feb;7(1):47-55 PMID: 3349904
  36. In vitro transcription of the Drosophila engrailed gene.
    Genes Dev. 1988 Jan;2(1):68-81 PMID: 3356339
  37. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts.
    Cell. 1988 Jun 3;53(5):699-711 PMID: 2897243
  38. GAL4 derivatives function alone and synergistically with mammalian activators in vitro.
    Cell. 1988 Aug 26;54(5):659-64 PMID: 3044607
  39. jockey, a mobile Drosophila element similar to mammalian LINEs, is transcribed from the internal promoter by RNA polymerase II.
    Cell. 1988 Aug 26;54(5):685-91 PMID: 2842063
  40. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates.
    J Biol Chem. 1979 Jul 10;254(13):6163-73 PMID: 447704
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.88.3.1024
Published
1991-02-01
Pages
1024-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC50947
Subset
IM
Grants
NIGMS NIH HHS · GM 41249 · United States
External Links
PubMed source DOI: 10.1073/pnas.88.3.1024 PMC Full Text View journal details
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