Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Jan;78(1):205–209. doi: 10.1073/pnas.78.1.205

Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.

S Tabor, C C Richardson
PMCID: PMC319020  PMID: 6454135

Abstract

The gene 4 protein of bacteriophage T7 recognizes specific sequences on single-stranded DNA and then catalyzes the synthesis of tetraribonucleotide primers complementary to the template. With phi X174 DNA as a template, the gene 4 protein enables T7 DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) to initiate DNA synthesis at 13 major sites. DNA sequence analysis of the 5' termini of the newly synthesized DNA shows the predominant recognition sequences for the gene 4 protein to be 3'-C-T-G-G-G-5' or 3'-C-T-G-G-T-5'; the products of synthesis at these sites are RNA primers having the sequences pppA-C-C-C or pppA-C-C-A. The gene 4 protein can also synthesize primers at the sequences 3'-C-T-G-G-AC-5' and 3'-C-T-G-T-N-5', although these sites are used less than 10% as frequently as the predominant sites. Comparison of the utilization of primer sites suggests that the gene 4 protein binds randomly to single-stranded DNA and then translocates along the DNA in a unidirectional 5'-to-3' direction with regard to the DNA strand in search of recognition sequences. Models are presented for the role of the gene 4 protein in the initiation of lagging-strand synthesis and in the initiation of DNA replication at the origin.

Full text

PDF
205

Images in this article

206Image
on p.206
207Image
on p.207

Selected References

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

  1. Adler S., Modrich P. T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits. J Biol Chem. 1979 Nov 25;254(22):11605–11614. [PubMed] [Google Scholar]
  2. Deutscher M. P. Synthesis and functions of the -C-C-A terminus of transfer RNA. Prog Nucleic Acid Res Mol Biol. 1973;13:51–92. doi: 10.1016/s0079-6603(08)60100-2. [DOI] [PubMed] [Google Scholar]
  3. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Purification and properties of the gene 4 protein of bacteriophage T7. J Biol Chem. 1975 Jul 25;250(14):5523–5529. [PubMed] [Google Scholar]
  4. Hutchison C. A., 3rd, Sinsheimer R. L. The process of infection with bacteriophage phi-X174. X. Mutations in a phi-X Lysis gene. J Mol Biol. 1966 Jul;18(3):429–447. doi: 10.1016/s0022-2836(66)80035-9. [DOI] [PubMed] [Google Scholar]
  5. Kolodner R., Masamune Y., LeClerc J. E., Richardson C. C. Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains. J Biol Chem. 1978 Jan 25;253(2):566–573. [PubMed] [Google Scholar]
  6. Kolodner R., Richardson C. C. Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1525–1529. doi: 10.1073/pnas.74.4.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kuhn B., Abdel-Monem M., Krell H., Hoffmann-Berling H. Evidence for two mechanisms for DNA unwinding catalyzed by DNA helicases. J Biol Chem. 1979 Nov 25;254(22):11343–11350. [PubMed] [Google Scholar]
  8. Liu C. C., Burke R. L., Hibner U., Barry J., Alberts B. Probing DNA replication mechanisms with the T4 bacteriophage in vitro system. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):469–487. doi: 10.1101/sqb.1979.043.01.053. [DOI] [PubMed] [Google Scholar]
  9. Mark D. F., Richardson C. C. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1976 Mar;73(3):780–784. doi: 10.1073/pnas.73.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. McMacken R., Ueda K., Kornberg A. Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4190–4194. doi: 10.1073/pnas.74.10.4190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Modrich P., Richardson C. C. Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J Biol Chem. 1975 Jul 25;250(14):5508–5514. [PubMed] [Google Scholar]
  13. Modrich P., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. J Biol Chem. 1975 Jul 25;250(14):5515–5522. [PubMed] [Google Scholar]
  14. Nossal N. G. RNA priming of DNA replication by bacteriophage T4 proteins. J Biol Chem. 1980 Mar 10;255(5):2176–2182. [PubMed] [Google Scholar]
  15. Ogawa T., Okazaki T. Discontinuous DNA replication. Annu Rev Biochem. 1980;49:421–457. doi: 10.1146/annurev.bi.49.070180.002225. [DOI] [PubMed] [Google Scholar]
  16. Okazaki R., Hirose S., Okazaki T., Ogawa T., Kurosawa Y. Assay of RNA-linked nascent DNA pieces with polynucleotide kinase. Biochem Biophys Res Commun. 1975 Feb 17;62(4):1018–1024. doi: 10.1016/0006-291x(75)90424-6. [DOI] [PubMed] [Google Scholar]
  17. Okazaki T., Kurosawa Y., Ogawa T., Seki T., Shinozaki K., Hirose S., Fujiyama A., Kohara Y., Machida Y., Tamanoid F. Structure and metabolism of the RNA primer in the discontinuous replication of prokaryotic DNA. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):203–219. doi: 10.1101/sqb.1979.043.01.026. [DOI] [PubMed] [Google Scholar]
  18. Richardson C. C., Romano L. J., Kolodner R., LeClerc J. E., Tamanoi F., Engler M. J., Dean F. B., Richardson D. S. Replication of bacteriophage T7 DNA by purified proteins. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):427–440. doi: 10.1101/sqb.1979.043.01.049. [DOI] [PubMed] [Google Scholar]
  19. Romano L. J., Richardson C. C. Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7. J Biol Chem. 1979 Oct 25;254(20):10483–10489. [PubMed] [Google Scholar]
  20. Romano L. J., Richardson C. C. Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7. J Biol Chem. 1979 Oct 25;254(20):10476–10482. [PubMed] [Google Scholar]
  21. Saito H., Tabor S., Tamanoi F., Richardson C. C. Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3917–3921. doi: 10.1073/pnas.77.7.3917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Coulson A. R., Friedmann T., Air G. M., Barrell B. G., Brown N. L., Fiddes J. C., Hutchison C. A., 3rd, Slocombe P. M., Smith M. The nucleotide sequence of bacteriophage phiX174. J Mol Biol. 1978 Oct 25;125(2):225–246. doi: 10.1016/0022-2836(78)90346-7. [DOI] [PubMed] [Google Scholar]
  23. Scherzinger E., Klotz G. Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins. Mol Gen Genet. 1975 Dec 1;141(3):233–249. doi: 10.1007/BF00341802. [DOI] [PubMed] [Google Scholar]
  24. Scherzinger E., Lanka E., Hillenbrand G. Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis. Nucleic Acids Res. 1977 Dec;4(12):4151–4163. doi: 10.1093/nar/4.12.4151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Scherzinger E., Lanka E., Morelli G., Seiffert D., Yuki A. Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. Eur J Biochem. 1977 Feb;72(3):543–558. doi: 10.1111/j.1432-1033.1977.tb11278.x. [DOI] [PubMed] [Google Scholar]
  26. Silver L. L., Nossal N. G. DNA replication by bacteriophage T4 proteins: role of the DNA-delay gene 61 in the chain-initiation reaction. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):489–494. doi: 10.1101/sqb.1979.043.01.054. [DOI] [PubMed] [Google Scholar]
  27. Tamanoi F., Saito H., Richardson C. C. Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication. Proc Natl Acad Sci U S A. 1980 May;77(5):2656–2660. doi: 10.1073/pnas.77.5.2656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weiss B., Live T. R., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. V. End group labeling and analysis of deoxyribonucleic acid containing single straned breaks. J Biol Chem. 1968 Sep 10;243(17):4530–4542. [PubMed] [Google Scholar]
  29. Wolfson J., Dressler D. Bacteriophage T7 DNA replication. An electron microscopic study of the growing point and the role of the T7 gene 4 protein in the formation of DNA fragments. J Biol Chem. 1979 Oct 25;254(20):10490–10495. [PubMed] [Google Scholar]
  30. Yarranton G. T., Das R. H., Gefter M. L. Enzyme-catalyzed DNA unwinding. Mechanism of action of helicase III. J Biol Chem. 1979 Dec 10;254(23):12002–12006. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES