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. 1984 Oct;81(19):5916–5920. doi: 10.1073/pnas.81.19.5916

Isolation of an active transcription initiation complex from HeLa cell-free extract.

H E Tolunay, L Yang, W F Anderson, B Safer
PMCID: PMC391829  PMID: 6592595

Abstract

A two-step procedure has been developed for the formation of RNA polymerase II transcription initiation and elongation complexes. Initiation complexes are rapidly formed in HeLa cell-free extract supplemented with a DNA template containing the adenovirus 2 major late promoter and ATP. Assembly of transcription components required for correct initiation is absolutely dependent on specific eukaryotic promoter sequences. Sarkosyl-sensitive transcription initiation complexes are rapidly converted to Sarkosyl-resistant elongation complexes when supplemented with the remaining nucleoside triphosphates. The 60S initiation complex can be extensively purified by glycerol gradient centrifugation and is easily separated from free RNA polymerase II and free DNA template. Recovery of this stable complex is greater than 90%. Specific transcription cannot be detected if the DNA template is subsequently added to gradient fractions containing HeLa cell-free extract components alone. This suggests that the DNA templates promote the specific assembly of RNA polymerase II and transcription factors required for accurate initiation. Since conversion of purified initiation complexes to elongation complexes can occur without additional HeLa cell components, the presence of transcription components required for initiation and elongation in a single complex is indicated.

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Selected References

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

  1. Ackerman S., Bunick D., Zandomeni R., Weinmann R. RNA polymerase II ternary transcription complexes generated in vitro. Nucleic Acids Res. 1983 Sep 10;11(17):6041–6064. doi: 10.1093/nar/11.17.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bunick D., Zandomeni R., Ackerman S., Weinmann R. Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts. Cell. 1982 Jul;29(3):877–886. doi: 10.1016/0092-8674(82)90449-4. [DOI] [PubMed] [Google Scholar]
  3. Davison B. L., Egly J. M., Mulvihill E. R., Chambon P. Formation of stable preinitiation complexes between eukaryotic class B transcription factors and promoter sequences. Nature. 1983 Feb 24;301(5902):680–686. doi: 10.1038/301680a0. [DOI] [PubMed] [Google Scholar]
  4. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  5. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  6. Gariglio P., Buss J., Green M. H. Sarkosyl activation of RNA polymerase activity in mitotic mouse cells. FEBS Lett. 1974 Aug 30;44(3):330–333. doi: 10.1016/0014-5793(74)81170-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Kedinger C., Gissinger F., Chambon P. Animal DNA-dependent RNA polymerases. Molecular structures and immunological properties of calf-thymus enzyme AI and of calf-thymus and rat-liver enzymes B. Eur J Biochem. 1974 May 15;44(2):421–436. doi: 10.1111/j.1432-1033.1974.tb03500.x. [DOI] [PubMed] [Google Scholar]
  9. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Matsui T., Segall J., Weil P. A., Roeder R. G. Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J Biol Chem. 1980 Dec 25;255(24):11992–11996. [PubMed] [Google Scholar]
  11. Safer B., Jagus R., Kemper W. M. Analysis of initiation factor function in highly fractionated and unfractionated reticulocyte lysate systems. Methods Enzymol. 1979;60:61–87. doi: 10.1016/s0076-6879(79)60008-3. [DOI] [PubMed] [Google Scholar]
  12. Samuels M., Fire A., Sharp P. A. Separation and characterization of factors mediating accurate transcription by RNA polymerase II. J Biol Chem. 1982 Dec 10;257(23):14419–14427. [PubMed] [Google Scholar]
  13. Shmookler R. J., Buss J., Green M. H. Properties of the polyoma virus transcription complex obtained from mouse nuclei. Virology. 1974 Jan;57(1):122–127. doi: 10.1016/0042-6822(74)90113-5. [DOI] [PubMed] [Google Scholar]
  14. Tolunay H. E., Yang L., Kemper W. M., Safer B., Anderson W. F. Homologous globin cell-free transcription system with comparison of heterologous factors. Mol Cell Biol. 1984 Jan;4(1):17–22. doi: 10.1128/mcb.4.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]

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