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Mol Cell Biol. 1995 Sep; 15(9): 4873–4883.
PMCID: PMC230733

RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.


Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adachi Y, Laemmli UK. Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. EMBO J. 1994 Sep 1;13(17):4153–4164. [PMC free article] [PubMed]
  • Adeniyi-Jones S, Romeo PH, Zasloff M. Generation of long read-through transcripts in vivo and in vitro by deletion of 3' termination and processing sequences in the human tRNAimet gene. Nucleic Acids Res. 1984 Jan 25;12(2):1101–1115. [PMC free article] [PubMed]
  • Adolph KW, Cheng SM, Laemmli UK. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977 Nov;12(3):805–816. [PubMed]
  • Almouzni G, Méchali M, Wolffe AP. Competition between transcription complex assembly and chromatin assembly on replicating DNA. EMBO J. 1990 Feb;9(2):573–582. [PMC free article] [PubMed]
  • Almouzni G, Wolffe AP. Nuclear assembly, structure, and function: the use of Xenopus in vitro systems. Exp Cell Res. 1993 Mar;205(1):1–15. [PubMed]
  • Almouzni G, Wolffe AP. Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo. Genes Dev. 1993 Oct;7(10):2033–2047. [PubMed]
  • Amberg DC, Goldstein AL, Cole CN. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 1992 Jul;6(7):1173–1189. [PubMed]
  • Barton MC, Emerson BM. Regulated expression of the beta-globin gene locus in synthetic nuclei. Genes Dev. 1994 Oct 15;8(20):2453–2465. [PubMed]
  • Barton MC, Madani N, Emerson BM. The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes. Genes Dev. 1993 Sep;7(9):1796–1809. [PubMed]
  • Bauer DW, Murphy C, Wu Z, Wu CH, Gall JG. In vitro assembly of coiled bodies in Xenopus egg extract. Mol Biol Cell. 1994 Jun;5(6):633–644. [PMC free article] [PubMed]
  • Bell P, Dabauvalle MC, Scheer U. In vitro assembly of prenucleolar bodies in Xenopus egg extract. J Cell Biol. 1992 Sep;118(6):1297–1304. [PMC free article] [PubMed]
  • Blow JJ, Laskey RA. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. [PubMed]
  • Carter KC, Taneja KL, Lawrence JB. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. [PMC free article] [PubMed]
  • Cox LS, Laskey RA. DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro. Cell. 1991 Jul 26;66(2):271–275. [PubMed]
  • Dabauvalle MC, Loos K, Scheer U. Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma. 1990 Dec;100(1):56–66. [PubMed]
  • Dargemont C, Schmidt-Zachmann MS, Kühn LC. Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus. J Cell Sci. 1995 Jan;108(Pt 1):257–263. [PubMed]
  • Dasso M, Nishitani H, Kornbluth S, Nishimoto T, Newport JW. RCC1, a regulator of mitosis, is essential for DNA replication. Mol Cell Biol. 1992 Aug;12(8):3337–3345. [PMC free article] [PubMed]
  • Doye V, Wepf R, Hurt EC. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. [PMC free article] [PubMed]
  • Dworetzky SI, Feldherr CM. Translocation of RNA-coated gold particles through the nuclear pores of oocytes. J Cell Biol. 1988 Mar;106(3):575–584. [PMC free article] [PubMed]
  • Elliott DJ, Stutz F, Lescure A, Rosbash M. mRNA nuclear export. Curr Opin Genet Dev. 1994 Apr;4(2):305–309. [PubMed]
  • Fabre E, Boelens WC, Wimmer C, Mattaj IW, Hurt EC. Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif. Cell. 1994 Jul 29;78(2):275–289. [PubMed]
  • Fang F, Newport JW. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell. 1991 Aug 23;66(4):731–742. [PubMed]
  • Featherstone C, Darby MK, Gerace L. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J Cell Biol. 1988 Oct;107(4):1289–1297. [PMC free article] [PubMed]
  • Feldherr CM, Akin D. Role of nuclear trafficking in regulating cellular activity. Int Rev Cytol. 1994;151:183–228. [PubMed]
  • Finlay DR, Forbes DJ. Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell. 1990 Jan 12;60(1):17–29. [PubMed]
  • Finlay DR, Meier E, Bradley P, Horecka J, Forbes DJ. A complex of nuclear pore proteins required for pore function. J Cell Biol. 1991 Jul;114(1):169–183. [PMC free article] [PubMed]
  • Forbes DJ. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. [PubMed]
  • Forbes DJ, Kirschner MW, Newport JW. Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs. Cell. 1983 Aug;34(1):13–23. [PubMed]
  • Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. [PubMed]
  • Gottesfeld JM, Wolf VJ, Dang T, Forbes DJ, Hartl P. Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component. Science. 1994 Jan 7;263(5143):81–84. [PubMed]
  • Hartl P, Gottesfeld J, Forbes DJ. Mitotic repression of transcription in vitro. J Cell Biol. 1993 Feb;120(3):613–624. [PMC free article] [PubMed]
  • Hartl P, Olson E, Dang T, Forbes DJ. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate. J Cell Biol. 1994 Feb;124(3):235–248. [PMC free article] [PubMed]
  • Hassan AB, Errington RJ, White NS, Jackson DA, Cook PR. Replication and transcription sites are colocalized in human cells. J Cell Sci. 1994 Feb;107(Pt 2):425–434. [PubMed]
  • Hirano T, Mitchison TJ. Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. J Cell Biol. 1993 Feb;120(3):601–612. [PMC free article] [PubMed]
  • Hozák P, Cook PR. Replication factories. Trends Cell Biol. 1994 Feb;4(2):48–52. [PubMed]
  • Hozák P, Jackson DA, Cook PR. Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle. J Cell Sci. 1994 Aug;107(Pt 8):2191–2202. [PubMed]
  • Huang S, Spector DL. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. [PubMed]
  • Jackson DA. The organization of replication centres in higher eukaryotes. Bioessays. 1990 Feb;12(2):87–89. [PubMed]
  • Jackson DA, Hassan AB, Errington RJ, Cook PR. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. [PMC free article] [PubMed]
  • Jarmolowski A, Boelens WC, Izaurralde E, Mattaj IW. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. [PMC free article] [PubMed]
  • Kadowaki T, Chen S, Hitomi M, Jacobs E, Kumagai C, Liang S, Schneiter R, Singleton D, Wisniewska J, Tartakoff AM. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J Cell Biol. 1994 Aug;126(3):649–659. [PMC free article] [PubMed]
  • Kamakaka RT, Bulger M, Kadonaga JT. Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 1993 Sep;7(9):1779–1795. [PubMed]
  • Kassavetis GA, Bartholomew B, Blanco JA, Johnson TE, Geiduschek EP. Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7308–7312. [PMC free article] [PubMed]
  • Kassavetis GA, Riggs DL, Negri R, Nguyen LH, Geiduschek EP. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes. Mol Cell Biol. 1989 Jun;9(6):2551–2566. [PMC free article] [PubMed]
  • Kornbluth S, Sebastian B, Hunter T, Newport J. Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14. Mol Biol Cell. 1994 Mar;5(3):273–282. [PMC free article] [PubMed]
  • Krug RM. The regulation of export of mRNA from nucleus to cytoplasm. Curr Opin Cell Biol. 1993 Dec;5(6):944–949. [PubMed]
  • Lang I, Scholz M, Peters R. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J Cell Biol. 1986 Apr;102(4):1183–1190. [PMC free article] [PubMed]
  • Laskey RA, Leno GH. Assembly of the cell nucleus. Trends Genet. 1990 Dec;6(12):406–410. [PubMed]
  • Laybourn PJ, Kadonaga JT. Threshold phenomena and long-distance activation of transcription by RNA polymerase II. Science. 1992 Sep 18;257(5077):1682–1685. [PubMed]
  • Lazarides E, Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. [PMC free article] [PubMed]
  • Leno GH, Laskey RA. The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts. J Cell Biol. 1991 Feb;112(4):557–566. [PMC free article] [PubMed]
  • Lewin B. Chromatin and gene expression: constant questions, but changing answers. Cell. 1994 Nov 4;79(3):397–406. [PubMed]
  • Logan K, Ackerman S. Effects of antibiotics on RNA polymerase III transcription. DNA. 1988 Sep;7(7):483–491. [PubMed]
  • Lohka MJ, Maller JL. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. [PMC free article] [PubMed]
  • Lohka MJ, Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. [PubMed]
  • Lohka MJ, Masui Y. Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol. 1984 Apr;98(4):1222–1230. [PMC free article] [PubMed]
  • Miake-Lye R, Kirschner MW. Induction of early mitotic events in a cell-free system. Cell. 1985 May;41(1):165–175. [PubMed]
  • Mills AD, Blow JJ, White JG, Amos WB, Wilcock D, Laskey RA. Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. J Cell Sci. 1989 Nov;94(Pt 3):471–477. [PubMed]
  • Méchali M, Harland RM. DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. Cell. 1982 Aug;30(1):93–101. [PubMed]
  • Newmeyer DD, Finlay DR, Forbes DJ. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2091–2102. [PMC free article] [PubMed]
  • Newmeyer DD, Forbes DJ. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. [PubMed]
  • Newmeyer DD, Lucocq JM, Bürglin TR, De Robertis EM. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. [PMC free article] [PubMed]
  • Newmeyer DD, Wilson KL. Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 1991;36:607–634. [PubMed]
  • Newport J, Spann T, Kanki J, Forbes D. The role of mitotic factors in regulating the timing of the midblastula transition in Xenopus. Cold Spring Harb Symp Quant Biol. 1985;50:651–656. [PubMed]
  • Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. [PubMed]
  • Newport J, Dunphy W. Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. J Cell Biol. 1992 Jan;116(2):295–306. [PMC free article] [PubMed]
  • Newport JW, Forbes DJ. The nucleus: structure, function, and dynamics. Annu Rev Biochem. 1987;56:535–565. [PubMed]
  • Newport J, Spann T. Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell. 1987 Jan 30;48(2):219–230. [PubMed]
  • Newport JW, Wilson KL, Dunphy WG. A lamin-independent pathway for nuclear envelope assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2247–2259. [PMC free article] [PubMed]
  • Pazin MJ, Kamakaka RT, Kadonaga JT. ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates. Science. 1994 Dec 23;266(5193):2007–2011. [PubMed]
  • Peterson RC, Doering JL, Brown DD. Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA. Cell. 1980 May;20(1):131–141. [PubMed]
  • Powers MA, Macaulay C, Masiarz FR, Forbes DJ. Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication. J Cell Biol. 1995 Mar;128(5):721–736. [PMC free article] [PubMed]
  • Schlenstedt G, Saavedra C, Loeb JD, Cole CN, Silver PA. The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):225–229. [PMC free article] [PubMed]
  • Sheehan MA, Mills AD, Sleeman AM, Laskey RA, Blow JJ. Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J Cell Biol. 1988 Jan;106(1):1–12. [PMC free article] [PubMed]
  • Smith DR, Jackson IJ, Brown DD. Domains of the positive transcription factor specific for the Xenopus 5S RNA gene. Cell. 1984 Jun;37(2):645–652. [PubMed]
  • Smythe C, Newport JW. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 1991;35:449–468. [PubMed]
  • Spector DL. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. [PubMed]
  • Sullivan KM, Busa WB, Wilson KL. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 1993 Jul 2;73(7):1411–1422. [PubMed]
  • Vigers GP, Lohka MJ. A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs. J Cell Biol. 1991 Feb;112(4):545–556. [PMC free article] [PubMed]
  • Wallrath LL, Lu Q, Granok H, Elgin SC. Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures. Bioessays. 1994 Mar;16(3):165–170. [PubMed]
  • Wansink DG, Schul W, van der Kraan I, van Steensel B, van Driel R, de Jong L. Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol. 1993 Jul;122(2):283–293. [PMC free article] [PubMed]
  • Wente SR, Blobel G. A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic. J Cell Biol. 1993 Oct;123(2):275–284. [PMC free article] [PubMed]
  • Wolffe AP. Replication timing and Xenopus 5S RNA gene transcription in vitro. Dev Biol. 1993 May;157(1):224–231. [PubMed]
  • Wolffe AP. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei. Nucleic Acids Res. 1989 Jan 25;17(2):767–780. [PMC free article] [PubMed]
  • Wolffe AP, Brown DD. Differential 5S RNA gene expression in vitro. Cell. 1987 Dec 4;51(5):733–740. [PubMed]
  • Workman JL, Buchman AR. Multiple functions of nucleosomes and regulatory factors in transcription. Trends Biochem Sci. 1993 Mar;18(3):90–95. [PubMed]
  • Xing Y, Johnson CV, Dobner PR, Lawrence JB. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. [PubMed]
  • Yan H, Newport J. An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. J Cell Biol. 1995 Apr;129(1):1–15. [PMC free article] [PubMed]

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