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. 1979 Dec 11;7(7):1851–1867. doi: 10.1093/nar/7.7.1851

Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping.

B K Chelm, E P Geiduschek
PMCID: PMC342352  PMID: 537912

Abstract

We describe a method for analyzing ternary transcription complexes, of RNA polymerase, DNA and nascent RNA32 chains, by agarose gel electrophoresis. When the RNA of such complexes is 32P-labelled, a simple comparison of the DNA fluorogram with an autoradiogram identifies transcriptionally active DNA molecules and restriction fragments in any mixture. Two limitations on the method are described: 1) retardation during electrophoresis of polymerase-DNA complexes relative to their conjugate bare NA fragments; 2) failure of very large ternary complexes to enter gels. The following potential applications of the method are surveyed: transcription unit (elongation) mapping, separation of RNA molecules in a mixture of transcripts, dinucleotide primer mapping and identification of preferred template conformations.

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Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  3. Blattner F. R., Dahlberg J. E. RNA synthesis startpoints in bacteriophage lambda: are the promoter and operator transcribed? Nat New Biol. 1972 Jun 21;237(77):227–232. doi: 10.1038/newbio237227a0. [DOI] [PubMed] [Google Scholar]
  4. Brooks T. L., Green M. H. The sv40 transcription complex. I. Effect of viral chromatin proteins on endogenous RNA polymerase activity. Nucleic Acids Res. 1977 Dec;4(12):4261–4277. doi: 10.1093/nar/4.12.4261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  6. Darlix J. L., Sentenac A., Fromageot P. Etude du RNA synthétisé in vitro. Biochim Biophys Acta. 1968 Sep 24;166(2):438–458. [PubMed] [Google Scholar]
  7. Downey K. M., So A. G. Studies on the kinetics of ribonucleic acid chain initiation and elongation. Biochemistry. 1970 Jun 9;9(12):2520–2525. doi: 10.1021/bi00814a019. [DOI] [PubMed] [Google Scholar]
  8. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  9. Duffy J. J., Geiduschek E. P. RNA polymerase from phage SP01-infected and uninfected Bacillus subtilis. J Biol Chem. 1975 Jun 25;250(12):4530–4541. [PubMed] [Google Scholar]
  10. Fuchse, Millette R. L., Zillig W., Walter G. Influence of salts on RNA synthesis by DNA-dependent RNA-polymerase from Escherichia coli. Eur J Biochem. 1967 Dec;3(2):183–193. doi: 10.1111/j.1432-1033.1967.tb19514.x. [DOI] [PubMed] [Google Scholar]
  11. Geiduschek E. P., Armelin M. C., Petrusek R., Bread C., Duffy J. J., Johnson G. Effects of the transciption inhibitory protein, TF1, on phage SP01 promoter complex formation and stability. J Mol Biol. 1977 Dec 25;117(4):825–842. doi: 10.1016/s0022-2836(77)80001-6. [DOI] [PubMed] [Google Scholar]
  12. Golomb M., Chamberlin M. A preliminary map of the major transcription units read by T7 RNA polymerase on the T7 and T3 bacteriophage chromosomes. Proc Natl Acad Sci U S A. 1974 Mar;71(3):760–764. doi: 10.1073/pnas.71.3.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hallick R. B., Lipper C., Richards O. C., Rutter W. J. Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis. Biochemistry. 1976 Jul 13;15(14):3039–3045. doi: 10.1021/bi00659a016. [DOI] [PubMed] [Google Scholar]
  14. Hoffman D. J., Niyogi S. K. RNA initiation with dinucleoside monophosphates during transcription of bacteriophage T4 DNA with RNA polymerase of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Feb;70(2):574–578. doi: 10.1073/pnas.70.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito J. Bacteriophage phi29 terminal protein: its association with the 5' termini of the phi29 genome. J Virol. 1978 Dec;28(3):895–904. doi: 10.1128/jvi.28.3.895-904.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ito J., Kawamura F., Duffy J. J. Susceptibility of non-thymine containing DNA to four bacterial restriction endonucleases. FEBS Lett. 1975 Jul 15;55(1):278–281. doi: 10.1016/0014-5793(75)81011-8. [DOI] [PubMed] [Google Scholar]
  17. Jones O. W., Berg P. Studies on the binding of RNA polymerase to polynucleotides. J Mol Biol. 1966 Dec 28;22(2):199–209. doi: 10.1016/0022-2836(66)90126-4. [DOI] [PubMed] [Google Scholar]
  18. Kawamura F., Ito J. Transcription of the genome of bacteriophage phi 29: isolation and mapping of the major early mRNA synthesized in vivo and in vitro. J Virol. 1977 Sep;23(3):562–577. doi: 10.1128/jvi.23.3.562-577.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knapp G., Beckmann J. S., Johnson P. F., Fuhrman S. A., Abelson J. Transcription and processing of intervening sequences in yeast tRNA genes. Cell. 1978 Jun;14(2):221–236. doi: 10.1016/0092-8674(78)90109-5. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Lilley D. M., Houghton M. The interaction of RNA polymerase II from wheat with supercoiled and linear plasmid templates. Nucleic Acids Res. 1979 Feb;6(2):507–523. doi: 10.1093/nar/6.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. MANDELL J. D., HERSHEY A. D. A fractionating column for analysis of nucleic acids. Anal Biochem. 1960 Jun;1:66–77. doi: 10.1016/0003-2697(60)90020-8. [DOI] [PubMed] [Google Scholar]
  23. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  24. Minkley E. G., Pribnow D. Transcription of the early region of bacteriophage T7: selective initiation with dinucleotides. J Mol Biol. 1973 Jun 25;77(2):255–277. doi: 10.1016/0022-2836(73)90335-5. [DOI] [PubMed] [Google Scholar]
  25. Otsuka A., Abelson J. The regulatory region of the biotin operon in Escherichia coli. Nature. 1978 Dec 14;276(5689):689–694. doi: 10.1038/276689a0. [DOI] [PubMed] [Google Scholar]
  26. Richardson J. P. Effects of supercoiling on transcription from bacteriophage PM2 deoxyribonucleic acid. Biochemistry. 1974 Jul 16;13(15):3164–3169. doi: 10.1021/bi00712a025. [DOI] [PubMed] [Google Scholar]
  27. Richardson J. P. The binding of RNA polymerase to DNA. J Mol Biol. 1966 Oct 28;21(1):83–114. doi: 10.1016/0022-2836(66)90081-7. [DOI] [PubMed] [Google Scholar]
  28. Robinson A. J., Younghusband H. B., Bellett A. J. A circula DNA-protein complex from adenoviruses. Virology. 1973 Nov;56(1):54–69. doi: 10.1016/0042-6822(73)90287-0. [DOI] [PubMed] [Google Scholar]
  29. Salas M., Mellado R. P., Viñuela E. Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29. J Mol Biol. 1978 Feb 25;119(2):269–291. doi: 10.1016/0022-2836(78)90438-2. [DOI] [PubMed] [Google Scholar]
  30. Seeburg P. H., Nüsslein C., Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd. Eur J Biochem. 1977 Mar 15;74(1):107–113. doi: 10.1111/j.1432-1033.1977.tb11372.x. [DOI] [PubMed] [Google Scholar]
  31. Sogo J. M., Inciarte M. R., Corral J., Viñuela E., Salas M. RNA polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage phi29. J Mol Biol. 1979 Feb 5;127(4):411–436. doi: 10.1016/0022-2836(79)90230-4. [DOI] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. Stahl S. J., Chamberlin M. J. An expanded transcriptional map of T7 bacteriophage. Reading of minor T7 promoter sites in vitro by Escherichia coli RNA polymerase. J Mol Biol. 1977 Jun 5;112(4):577–601. doi: 10.1016/s0022-2836(77)80165-4. [DOI] [PubMed] [Google Scholar]
  34. Stonington O. G., Pettijohn D. E. The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc Natl Acad Sci U S A. 1971 Jan;68(1):6–9. doi: 10.1073/pnas.68.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  36. Todd R. D., Garrard W. T. Two-dimensional electrophoretic analysis of polynucleosomes. J Biol Chem. 1977 Jul 10;252(13):4729–4738. [PubMed] [Google Scholar]
  37. Williams R. C. Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2311–2315. doi: 10.1073/pnas.74.6.2311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zillig W., Zechel K., Halbwachs H. J. A new method of large scale preparation of highly purified DNA-dependent RNA-polymerase from E. coli. Hoppe Seylers Z Physiol Chem. 1970 Feb;351(2):221–224. doi: 10.1515/bchm2.1970.351.1.221. [DOI] [PubMed] [Google Scholar]

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