Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Sep 10;11(17):6041–6064. doi: 10.1093/nar/11.17.6041

RNA polymerase II ternary transcription complexes generated in vitro.

S Ackerman, D Bunick, R Zandomeni, R Weinmann
PMCID: PMC326334  PMID: 6193489

Abstract

Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation.

Full text

PDF
6041

Images in this article

6045Image
on p.6045
6047Image
on p.6047
6048Image
on p.6048
6049Image
on p.6049
6051Image
on p.6051
6052Image
on p.6052
6055Image
on p.6055

Selected References

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

  1. Baker C. C., Ziff E. B. Promoters and heterogeneous 5' termini of the messenger RNAs of adenovirus serotype 2. J Mol Biol. 1981 Jun 25;149(2):189–221. doi: 10.1016/0022-2836(81)90298-9. [DOI] [PubMed] [Google Scholar]
  2. Ballario P., Buongiorno-Nardelli M., Carnevali F., Di Mauro E., Pedone F. Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 micron DNA. Nucleic Acids Res. 1981 Aug 25;9(16):3959–3978. doi: 10.1093/nar/9.16.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beard P. Mobility of histones on the chromosome of simian virus 40. Cell. 1978 Nov;15(3):955–967. doi: 10.1016/0092-8674(78)90279-9. [DOI] [PubMed] [Google Scholar]
  4. Beard P., Nyfeler K. Transcription of Simian Virus 40 chromosomes in an extract of HeLa cells. EMBO J. 1982;1(1):9–14. doi: 10.1002/j.1460-2075.1982.tb01116.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Carnevali F., Caserta M., Di Mauro E. In vitro transcription by purified yeast RNA polymerase II. Coarse promoter mapping on homologous cloned genes. Nucleic Acids Res. 1982 May 25;10(10):3195–3209. doi: 10.1093/nar/10.10.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chelm B. K., Geiduschek E. P. Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping. Nucleic Acids Res. 1979 Dec 11;7(7):1851–1867. doi: 10.1093/nar/7.7.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cochet-Meilhac M., Chambon P. Animal DNA-dependent RNA polymerases. 11. Mechanism of the inhibition of RNA polymerases B by amatoxins. Biochim Biophys Acta. 1974 Jun 27;353(2):160–184. doi: 10.1016/0005-2787(74)90182-8. [DOI] [PubMed] [Google Scholar]
  11. Crepin M., Triadou P., Lelong J. C., Gros F. Identification of transcription initiation sites for bacterial RNA polymerase and eukaryotic RNA polymerase B on the 5' end of the mouse beta-Globin gene. Eur J Biochem. 1981 Aug;118(2):371–377. doi: 10.1111/j.1432-1033.1981.tb06412.x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Dynan W. S., Burgess R. R. In vitro transcription by wheat germ RNA polymerase II. Initiation of RNA synthesis on relaxed, closed circular template. J Biol Chem. 1981 Jun 10;256(11):5866–5873. [PubMed] [Google Scholar]
  14. 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]
  15. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gamper H. B., Hearst J. E. A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes. Cell. 1982 May;29(1):81–90. doi: 10.1016/0092-8674(82)90092-7. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gralla J. D., Carpousis A. J., Stefano J. E. Productive and abortive initiation of transcription in vitro at the lac UV5 promoter. Biochemistry. 1980 Dec 9;19(25):5864–5869. doi: 10.1021/bi00566a031. [DOI] [PubMed] [Google Scholar]
  20. Hansen U. M., McClure W. R. Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. II. Release of sigma from ternary complexes. J Biol Chem. 1980 Oct 25;255(20):9564–9570. [PubMed] [Google Scholar]
  21. Kadesch T. R., Rosenberg S., Chamberlin M. J. Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of binding at bacteriophage T7 promoter A1 using a template competition assay. J Mol Biol. 1982 Feb 15;155(1):1–29. doi: 10.1016/0022-2836(82)90489-2. [DOI] [PubMed] [Google Scholar]
  22. Lescure B., Bennetzen J., Sentenac A. In vitro transcription of the yeast alcohol dehydrogenase I gene by homologous RNA polymerase B (II). Selective initiation and discontinuous elongation on a supercoiled template. J Biol Chem. 1981 Nov 10;256(21):11018–11024. [PubMed] [Google Scholar]
  23. Luse D. S., Haynes J. R., VanLeeuwen D., Schon E. A., Cleary M. L., Shapiro S. G., Lingrel J. B., Roeder R. G. Transcription of the beta-like globin genes and pseudogenes of the goat in a cell-free system. Nucleic Acids Res. 1981 Sep 11;9(17):4339–4354. doi: 10.1093/nar/9.17.4339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  27. Melançon P., Burgess R. R., Record M. T., Jr Nitrocellulose filter binding studies of the interactions of Escherichia coli RNA polymerase holoenzyme with deoxyribonucleic acid restriction fragments: evidence for multiple classes of nonpromoter interactions, some of which display promoter-like properties. Biochemistry. 1982 Aug 31;21(18):4318–4331. doi: 10.1021/bi00261a022. [DOI] [PubMed] [Google Scholar]
  28. Rosenberg S., Kadesch T. R., Chamberlin M. J. Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of the rate of open complex formation at T7 promoter A. J Mol Biol. 1982 Feb 15;155(1):31–51. doi: 10.1016/0022-2836(82)90490-9. [DOI] [PubMed] [Google Scholar]
  29. Sassone-Corsi P., Corden J., Kédinger C., Chambon P. Promotion of specific in vitro transcription by excised "TATA" box sequences inserted in a foreign nucleotide environment. Nucleic Acids Res. 1981 Aug 25;9(16):3941–3958. doi: 10.1093/nar/9.16.3941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sinha S. N., Hellwig R. J., Allison D. P., Niyogi S. K. Conversion of simian virus 40 DNA to ordered nucleoprotein structures by extracts that direct accurate initiation by eukaryotic RNA polymerase II. Nucleic Acids Res. 1982 Sep 25;10(18):5533–5552. doi: 10.1093/nar/10.18.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsai S. Y., Tsai M. J., Kops L. E., Minghetti P. P., O'Malley B. W. Transcription factors from oviduct and HeLa cells are similar. J Biol Chem. 1981 Dec 25;256(24):13055–13059. [PubMed] [Google Scholar]
  33. Vaisius A. C., Wieland T. Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin. Biochemistry. 1982 Jun 22;21(13):3097–3101. doi: 10.1021/bi00256a010. [DOI] [PubMed] [Google Scholar]
  34. Weil P. A., Luse D. S., Segall J., Roeder R. G. 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):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]
  35. Zandomeni R., Mittleman B., Bunick D., Ackerman S., Weinmann R. Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription. Proc Natl Acad Sci U S A. 1982 May;79(10):3167–3170. doi: 10.1073/pnas.79.10.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES