Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1969 Nov;115(3):353–361. doi: 10.1042/bj1150353

Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

John O Bishop 1, Forbes W Robertson 1
PMCID: PMC1185113  PMID: 4901068

Abstract

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

Full text

PDF
355

Selected References

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

  1. Bishop J. O., Robertson F. W., Burns J. A., Melli M. Methods for the analysis of deoxyribonucleic acid-ribonucleic acid hybridization data. Biochem J. 1969 Nov;115(3):361–370. doi: 10.1042/bj1150361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop J. O. The effect of genetic complexity on the time-course of ribonucleic acid-deoxyribonucleic acid hybridization. Biochem J. 1969 Aug;113(5):805–811. doi: 10.1042/bj1130805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop J. O. Transcription of denatured T4 DNA. Biochim Biophys Acta. 1969 Feb 18;174(2):636–652. doi: 10.1016/0005-2787(69)90293-7. [DOI] [PubMed] [Google Scholar]
  4. Bolle A., Epstein R. H., Salser W., Geiduschek E. P. Transcription during bacteriophage T4 development: synthesis and relative stability of early and late RNA. J Mol Biol. 1968 Feb 14;31(3):325–348. doi: 10.1016/0022-2836(68)90413-0. [DOI] [PubMed] [Google Scholar]
  5. Bremer H., Konrad M., Bruner R. Capacity of T4 DNA to serve as template for purified Escerichia coli RNA polymerase. J Mol Biol. 1966 Mar;16(1):104–117. doi: 10.1016/s0022-2836(66)80266-8. [DOI] [PubMed] [Google Scholar]
  6. Cohen S. N., Hurwitz J. Transcription of complementary strands of phage lambda-DNA in vivo and in vitro. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1759–1766. doi: 10.1073/pnas.57.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colvill A. J., Kanner L. C., Tocchini-Valentini G. P., Sarnat M. T., Geiduschek E. P. Asymmetric RNA synthesis in vitro: heterologous DNA-enzyme systems; E. coli RNA polymerase. Proc Natl Acad Sci U S A. 1965 May;53(5):1140–1147. doi: 10.1073/pnas.53.5.1140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FOX C. F., WEISS S. B. ENZYMATIC SYNTHESIS OF RIBONUCLEIC ACID. II. PROPERTIES OF THE DEOXYRIBONUCLEIC ACID-PRIMED REACTION WITH MICROCOCCUS LYSODEIKTICUS RIBONUCLEIC ACID POLYMERASE. J Biol Chem. 1964 Jan;239:175–185. [PubMed] [Google Scholar]
  9. FRASER D., JERREL E. A. The amino acid composition of T3 bacteriophage. J Biol Chem. 1953 Nov;205(1):291–295. [PubMed] [Google Scholar]
  10. FURTH J. J., HURWITZ J., GOLDMANN M. The directing role of DNA in RNA synthesis. Biochem Biophys Res Commun. 1961 Apr 7;4:362–367. doi: 10.1016/0006-291x(61)90219-4. [DOI] [PubMed] [Google Scholar]
  11. GEIDUSCHEK E. P., MOOHR J. W., WEISS S. B. The secondary structure of complementary RNA. Proc Natl Acad Sci U S A. 1962 Jun 15;48:1078–1086. doi: 10.1073/pnas.48.6.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GEIDUSCHEK E. P., NAKAMOTO T., WEISS S. B. The enzymatic synthesis of RNA: complementary interaction with DNA. Proc Natl Acad Sci U S A. 1961 Sep 15;47:1405–1415. doi: 10.1073/pnas.47.9.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geiduschek E. P., Snyder L., Colvill A. J., Sarnat M. Selective synthesis of T-even bacteriophage early messenger in vitro. J Mol Biol. 1966 Aug;19(2):541–547. doi: 10.1016/s0022-2836(66)80021-9. [DOI] [PubMed] [Google Scholar]
  14. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  15. HALL B. D., NYGAARD A. P., GREEN M. H. CONTROL OF T2-SPECIFIC RNA SYNTHESIS. J Mol Biol. 1964 Jul;9:143–153. doi: 10.1016/s0022-2836(64)80096-6. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. KHESIN R. B., GORLENKO Zh M., SHEMIAKIN M. F., BASS Ia, PROZOROV A. A. SVIAZ' MEZHDU SINTEZOM BELKOV I REGULIATSIEI OBRAZOVANIIA INFORMATSIONNYKH RNK V KLETKAKH E. COLI B PRI RAZVITII FAGA TS. Biokhimiia. 1963 Nov-Dec;28:1070–1086. [PubMed] [Google Scholar]
  18. 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]
  19. Maitra U., Nakata Y., Hurwitz J. The role of deoxyribonucleic acid in ribonucleic acid synthesis. XIV. A study of the initiation of ribonucleic acid synthesis. J Biol Chem. 1967 Nov 10;242(21):4908–4918. [PubMed] [Google Scholar]
  20. NAKAMOTO T., FOX C. F., WEISS S. B. ENZYMATIC SYNTHESIS OF RIBONUCLEIC ACID. I. PREPARATION OF RIBONUCLEIC ACID POLYMERASE FROM EXTRACTS OF MICROCOCCUS LYSODEIKTICUS. J Biol Chem. 1964 Jan;239:167–174. [PubMed] [Google Scholar]
  21. ROBINSON W. S., HSU W. I., FOX C. F., WEISS S. B. ENZYMATIC SYNTHESIS OF RIBONUCLEIC ACID. IV. THE DEOXYRIBONUCLEIC ACID-DIRECTED SYNTHESIS OF COMPLEMENTARY CYTOPLASMIC RIBONUCLEIC ACID COMPONENTS. J Biol Chem. 1964 Sep;239:2944–2951. [PubMed] [Google Scholar]
  22. Steck T. L., Caicuts M. J., Wilson R. G. The influence of divalent cations on the activity of the ribonucleic acid polymerase of Micrococcus lysodeikticus. J Biol Chem. 1968 May 25;243(10):2769–2778. [PubMed] [Google Scholar]
  23. Weiss S. B., Nakamoto T. THE ENZYMATIC SYNTHESIS OF RNA: NEAREST-NEIGHBOR BASE FREQUENCIES. Proc Natl Acad Sci U S A. 1961 Sep;47(9):1400–1405. doi: 10.1073/pnas.47.9.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES