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. 1974 Oct;1(10):1305–1316. doi: 10.1093/nar/1.10.1305

A simplified method for study of RNA conformation - reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite

LaDonne H Schulman 1, Robert Shapiro 1, David CF Law 1, Judith B Louis 1
PMCID: PMC344352  PMID: 10793691

Abstract

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNAfMet in solution. The procedure involves conversion of cytosine residues to N4-[14C]methylcytosines by treatment with 14CH3NH2 and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces 14C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNAfMet has been found to contain only six reactive cytosines: C1, C16, C17, C35, C75 and C76. In addition, slow reaction at Cm33 was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNAfMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.

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Selected References

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

  1. Burton K. Conversion of 4-thiouridine residues in tRNA into radioactive N-4-methylcytidine for sequence analysis. FEBS Lett. 1970 Jan 26;6(2):77–79. doi: 10.1016/0014-5793(70)80005-9. [DOI] [PubMed] [Google Scholar]
  2. Chang S. E. Selective modification of cytidine and uridine residues in Escherichia coli formylmethionine transfer ribonucleic acid. J Mol Biol. 1973 Apr 15;75(3):533–547. doi: 10.1016/0022-2836(73)90459-2. [DOI] [PubMed] [Google Scholar]
  3. Cramer F. Three-dimensional structure of tRNA. Prog Nucleic Acid Res Mol Biol. 1971;11:391–421. doi: 10.1016/s0079-6603(08)60333-5. [DOI] [PubMed] [Google Scholar]
  4. Dube S. K., Marcker K. A., Clark B. F., Cory S. Nucleotide sequence of N-formyl-methionyl-transfer RNA. Nature. 1968 Apr 20;218(5138):232–233. doi: 10.1038/218232a0. [DOI] [PubMed] [Google Scholar]
  5. Egan B. Z., Weiss J. F., Kelmers A. D. Separation and comparison of primary structures of three formylmethionine tRNAs from E. coli K-12 MO. Biochem Biophys Res Commun. 1973 Nov 16;55(2):320–327. doi: 10.1016/0006-291x(73)91090-5. [DOI] [PubMed] [Google Scholar]
  6. Favre A., Yaniv M., Michelson A. M. The photochemistry of 4-thiouridine in Escherichia coli t-RNA Vał1. Biochem Biophys Res Commun. 1969 Oct 8;37(2):266–271. doi: 10.1016/0006-291x(69)90729-3. [DOI] [PubMed] [Google Scholar]
  7. Fellner P., Ehresmann C., Stiegler P., Ebel J. P. Partial nucleotide sequence of 16S ribosomal RNA from E. coli. Nat New Biol. 1972 Sep 6;239(88):1–5. [PubMed] [Google Scholar]
  8. Goddard J. P., Schulman L. H. Conversion of exposed cytidine residues to uridine residues in Escherichia coli formylmethionine transfer ribonucleic acid. J Biol Chem. 1972 Jun 25;247(12):3864–3867. [PubMed] [Google Scholar]
  9. Hayatsu H., Inoue M. The oxygen-mediated reaction between 4-thiouracil derivatives and bi- sulfite. Isolation and characterization of 1-methyluracil 4-thiosulfate as an intermediate in the formation of 1-methyluracil-4-sulfonate. J Am Chem Soc. 1971 May 5;93(9):2301–2306. doi: 10.1021/ja00738a033. [DOI] [PubMed] [Google Scholar]
  10. Hayatsu H., Yano M. Permanganate oxidation of 4-thiouracil derivatives. Isolation and properties of 1-substituted-2-pyrimidone-4-sulfonates. Tetrahedron Lett. 1969 Feb;(9):755–758. doi: 10.1016/s0040-4039(01)87801-1. [DOI] [PubMed] [Google Scholar]
  11. Min Jou W., Haegeman G., Ysebaert M., Fiers W. Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein. Nature. 1972 May 12;237(5350):82–88. doi: 10.1038/237082a0. [DOI] [PubMed] [Google Scholar]
  12. Reeves R. H., Imura N., Schwam H., Weiss G. B., Schulman L. H., Chambers R. W. Transfer RNA, I. Isolation and characterization of a new yeast alanine transfer RNA. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1450–1457. doi: 10.1073/pnas.60.4.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schulman L. H. Structure and function of Escherichia coli formylmethionine transfer RNA. II. Effect of modification of guanosine residues on aminoacyl synthetase recognition. J Mol Biol. 1971 May 28;58(1):117–131. doi: 10.1016/0022-2836(71)90236-1. [DOI] [PubMed] [Google Scholar]
  14. Shapiro R., Law D. C., Weisgras J. M. A new chemical probe for single-stranded RNA. Biochem Biophys Res Commun. 1972 Oct 17;49(2):358–363. doi: 10.1016/0006-291x(72)90418-4. [DOI] [PubMed] [Google Scholar]
  15. Shapiro R., Weisgras J. M. Bisulfite-catalyzed transamination of cytosine and cytidine. Biochem Biophys Res Commun. 1970 Aug 24;40(4):839–843. doi: 10.1016/0006-291x(70)90979-4. [DOI] [PubMed] [Google Scholar]
  16. Ziff E. B., Fresco J. R. Chemical transformation of 4-thiouracil nucleosides to uracil and cytosine counterparts. J Am Chem Soc. 1968 Dec 18;90(26):7338–7342. doi: 10.1021/ja01028a027. [DOI] [PubMed] [Google Scholar]

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