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. 1972 Jun;69(6):1387–1391. doi: 10.1073/pnas.69.6.1387

Ribosomal RNA Synthesis In Vitro: A Protein-DNA Complex from Bacillus subtilis Active in Initiation of Transcription

Jay A Winsten †,*, P C Huang *,‡
PMCID: PMC426708  PMID: 4624758

Abstract

A protein-DNA transcription complex, isolated from logarithmic-phase cells of Bacillus subtilis, is active in the initiation of new rounds of RNA synthesis in vitro. Transcription directed by the endogenous DNA of the complex is sensitive to inhibitors of initiation. In addition, the effect of addition of a competing template indicates that RNA polymerase in the complex is bound to endogenous DNA in a dissociable, preinitiation state. Hybridization-competition analysis of the RNA product obtained from the in vitro reaction indicates that it is greatly enriched for ribosomal RNA sequences. This initiating complex should be useful for the study of regulation of expression of ribosomal RNA genes.

Keywords: rifampicin, heparin, hybridization, membrane fraction

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Selected References

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  1. BERNS K. I., THOMAS C. A., Jr ISOLATION OF HIGH MOLECULAR WEIGHT DNA FROM HEMOPHILUS INFLUENZAE. J Mol Biol. 1965 Mar;11:476–490. doi: 10.1016/s0022-2836(65)80004-3. [DOI] [PubMed] [Google Scholar]
  2. BREMER H., KONRAD M. W. A COMPLEX OF ENZYMATICALLY SYNTHESIZED RNA AND TEMPLATE DNA. Proc Natl Acad Sci U S A. 1964 May;51:801–808. doi: 10.1073/pnas.51.5.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43–46. doi: 10.1038/221043a0. [DOI] [PubMed] [Google Scholar]
  4. Chambon P., DuPraw E. J., Kornberg A. Biochemical studies of bacterial sporulation and germination. IX. Ribonucleic acid and deoxyribonucleic acid polymerases in nuclear fractions of vegetative cells and spores of Bacillus megaterium. J Biol Chem. 1968 Oct 10;243(19):5101–5109. [PubMed] [Google Scholar]
  5. Hussey C., Losick R., Sonenshein A. L. Ribosomal RNA synthesis is turned off during sporulation of Bacillus subtilis. J Mol Biol. 1971 Apr 14;57(1):59–70. doi: 10.1016/0022-2836(71)90119-7. [DOI] [PubMed] [Google Scholar]
  6. INMAN R. B., BALDWIN R. L. Helix-random coil transitions in synthetic DNAs of alternating sequence. J Mol Biol. 1962 Aug;5:172–184. doi: 10.1016/s0022-2836(62)80082-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Lazzarini R. A., Dahlberg A. E. The control of ribonucleic acid synthesis during amino acid deprivation in Escherichia coli. J Biol Chem. 1971 Jan 25;246(2):420–429. [PubMed] [Google Scholar]
  10. Lazzarini R. A. Differences in lysine-sRNA from spore and vegetative cells of Bacillus subtillis. Proc Natl Acad Sci U S A. 1966 Jul;56(1):185–190. doi: 10.1073/pnas.56.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mizuno S., Whiteley H. R. Nuclear fraction of Bacillus subtilis as a template for ribonucleic acid synthesis. J Bacteriol. 1968 Apr;95(4):1221–1237. doi: 10.1128/jb.95.4.1221-1237.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. NYGAARD A. P., HALL B. D. A method for the detection of RNA-DNA complexes. Biochem Biophys Res Commun. 1963 Jul 18;12:98–104. doi: 10.1016/0006-291x(63)90242-0. [DOI] [PubMed] [Google Scholar]
  13. Oishi M., Sueoka N. Location of genetic loci of ribosomal RNA on Bacillus subtilis chromosome. Proc Natl Acad Sci U S A. 1965 Aug;54(2):483–491. doi: 10.1073/pnas.54.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pettijohn D. E., Clarkson K., Kossman C. R., Stonington O. G. Synthesis of ribosomal RNA on a protein-DNA complex isolated from bacteria: a comparison of ribosomal RNA synthesis in vitro and in vivo. J Mol Biol. 1970 Sep 14;52(2):281–300. doi: 10.1016/0022-2836(70)90031-8. [DOI] [PubMed] [Google Scholar]
  15. Pettijohn D. E., Stonington O. G., Kossman C. R. Chain termination of ribosomal RNA synthesis in vitro. Nature. 1970 Oct 17;228(5268):235–239. doi: 10.1038/228235a0. [DOI] [PubMed] [Google Scholar]
  16. Pettijohn D. E. Transcription on an RNA polymerase-DNA complex from E. coli: evidence for in vitro synthesis of ribosomal RNA. Biochem Biophys Res Commun. 1969 Feb 21;34(4):541–547. doi: 10.1016/0006-291x(69)90416-1. [DOI] [PubMed] [Google Scholar]
  17. Salser W., Janin J., Levinthal C. Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli. J Mol Biol. 1968 Jan 28;31(2):237–266. doi: 10.1016/0022-2836(68)90442-7. [DOI] [PubMed] [Google Scholar]
  18. Smith I., Dubnau D., Morrell P., Marmur J. Chromosomal location of DNA base sequences complementary to transfer RNA and to 5 s, 16 s and 23 s ribosomal RNA in Bacillus subtilis. J Mol Biol. 1968 Apr 14;33(1):123–140. doi: 10.1016/0022-2836(68)90285-4. [DOI] [PubMed] [Google Scholar]
  19. Travers A. A., Kamen R. I., Schleif R. F. Factor necessary for ribosomal RNA synthesis. Nature. 1970 Nov 21;228(5273):748–751. doi: 10.1038/228748a0. [DOI] [PubMed] [Google Scholar]
  20. Walter G., Zillig W., Palm P., Fuchs E. Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem. 1967 Dec;3(2):194–201. doi: 10.1111/j.1432-1033.1967.tb19515.x. [DOI] [PubMed] [Google Scholar]
  21. Wells R. D., Larson J. E. Studies on the binding of actinomycin D to DNA and DNA model polymers. J Mol Biol. 1970 Apr 28;49(2):319–342. doi: 10.1016/0022-2836(70)90248-2. [DOI] [PubMed] [Google Scholar]
  22. YANKOFSKY S. A., SPIEGELMAN S. The identification of the ribosomal RNA cistron by sequence complementarity. II. Saturation of and competitive interaction at the RNA cistron. Proc Natl Acad Sci U S A. 1962 Aug;48:1466–1472. doi: 10.1073/pnas.48.8.1466. [DOI] [PMC free article] [PubMed] [Google Scholar]

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