Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1979 Nov 10;7(5):1233–1246. doi: 10.1093/nar/7.5.1233

A convenient sequencing method for 5' protein-linked RNAs.

A Nomoto, N Imura
PMCID: PMC342299  PMID: 229466

Abstract

A convenient nucleotide sequencing method for 5' end protein-linked RNAs was developed. Genome of LSc, 2ab poliovirus, which has a protein (VPg) covalently linked to the 5' terminus, was labelled with 125I Bolton and Hunter reagent after proteinase K treatment. No sign of labelling of nucleotide moiety in the genome with the reagent was detected. A labelled oligo peptide-linked ribonuclease T1 fragment was obtained from the 5' end of the genome. Analysis of the complex by two dimensional gel electrophoresis after partial alkali digestion or by the nucleotide sequencing method of Donis-Keller et al. (1) revealed that LSc, 2ab strain genome had an identical 5' end structure to that of Mahoney strain genome, that is, VPg-pUpUpApApApApCpApGp. Our results have shown that this labelling method is useful for analysis of 5' end sequence of RNAs linked to protein at the 5' termini.

Full text

PDF

Images in this article

Selected References

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

  1. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burroughs J. N., Brown F. Presence of a covalently linked protein on calicivirus RNA. J Gen Virol. 1978 Nov;41(2):443–446. doi: 10.1099/0022-1317-41-2-443. [DOI] [PubMed] [Google Scholar]
  3. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Doyle M., Holland J. J. Virus-induced interference in heterologously infected HeLa cells. J Virol. 1972 Jan;9(1):22–28. doi: 10.1128/jvi.9.1.22-28.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ehrenfeld E., Lund H. Untranslated vesicular stomatitis virus messenger RNA after poliovirus infection. Virology. 1977 Jul 15;80(2):297–308. doi: 10.1016/s0042-6822(77)80006-8. [DOI] [PubMed] [Google Scholar]
  6. Fiszman M., Bucchini D., Girard M. Purification of the Sabin strain of poliovirus type I through treatment with sarkozyl. J Virol. 1971 May;7(5):687–689. doi: 10.1128/jvi.7.5.687-689.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flanegan J. B., Petterson R. F., Ambros V., Hewlett N. J., Baltimore D. Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate RNAs of poliovirus. Proc Natl Acad Sci U S A. 1977 Mar;74(3):961–965. doi: 10.1073/pnas.74.3.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Golini F., Nomoto A., Wimmer E. The genome-linked protein of picornaviruses. IV. Difference in the VPg's of encephalomyocarditis virus and poliovirus as evidence that the genome-linked proteins are virus-coded. Virology. 1978 Aug;89(1):112–118. doi: 10.1016/0042-6822(78)90045-4. [DOI] [PubMed] [Google Scholar]
  9. Harris T. J., Dunn J. J., Wimmer E. Identification of specific fragments containing the 5' end of poliovirus RNA after ribonuclease III digestion. Nucleic Acids Res. 1978 Nov;5(11):4039–4054. doi: 10.1093/nar/5.11.4039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaufmann Y., Goldstein E., Penman S. Poliovirus-induced inhibition of polypeptide initiation in vitro on native polyribosomes. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1834–1838. doi: 10.1073/pnas.73.6.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klootwijk J., Klein I., Zabel P., van Kammen A. Cowpea mosaic virus RNAs have neither m7GpppN ... nor mono-, di- or triphosphates at their 5' ends. Cell. 1977 May;11(1):73–82. doi: 10.1016/0092-8674(77)90318-x. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lee Y. F., Kitamura N., Nomoto A., Wimmer E. Sequence studies of poliovirus RNA. IV. Nucleotide sequence complexities of poliovirus type 1, type 2 and two type 1 defective interfering particles RNAs, and fingerprint of the poliovirus type 3 genome. J Gen Virol. 1979 Aug;44(2):311–322. doi: 10.1099/0022-1317-44-2-311. [DOI] [PubMed] [Google Scholar]
  14. Lee Y. F., Nomoto A., Detjen B. M., Wimmer E. A protein covalently linked to poliovirus genome RNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):59–63. doi: 10.1073/pnas.74.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leibowitz R., Penman S. Regulation of protein synthesis in HeLa cells. 3. Inhibition during poliovirus infection. J Virol. 1971 Nov;8(5):661–668. doi: 10.1128/jvi.8.5.661-668.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nomoto A., Detjen B., Pozzatti R., Wimmer E. The location of the polio genome protein in viral RNAs and its implication for RNA synthesis. Nature. 1977 Jul 21;268(5617):208–213. doi: 10.1038/268208a0. [DOI] [PubMed] [Google Scholar]
  17. Nomoto A., Kitamura N., Golini F., Wimmer E. The 5'-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5345–5349. doi: 10.1073/pnas.74.12.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rensing U. F., Schoenmakers G. G. A sequence of 50 nucleotides from coliphage R17 RNA. Eur J Biochem. 1973 Feb 15;33(1):8–18. doi: 10.1111/j.1432-1033.1973.tb02648.x. [DOI] [PubMed] [Google Scholar]
  19. Rose J. K., Trachsel H., Leong K., Baltimore D. Inhibition of translation by poliovirus: inactivation of a specific initiation factor. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2732–2736. doi: 10.1073/pnas.75.6.2732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rothberg P. G., Harris T. J., Nomoto A., Wimmer E. O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4868–4872. doi: 10.1073/pnas.75.10.4868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sangar D. V., Rowlands D. J., Harris T. J., Brown F. Protein covalently linked to foot-and-mouth disease virus RNA. Nature. 1977 Aug 18;268(5621):648–650. doi: 10.1038/268648a0. [DOI] [PubMed] [Google Scholar]
  22. Shafritz D. A., Weinstein J. A., Safer B., Merrick W. C., Weber L. A., Hickey E. D., Baglioni C. Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3. Nature. 1976 May 27;261(5558):291–294. doi: 10.1038/261291a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES