Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1979 Oct 10;7(3):765–779. doi: 10.1093/nar/7.3.765

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.

J G Tompson, P F Agris
PMCID: PMC328054  PMID: 388347

Abstract

Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.

Full text

PDF
779

Selected References

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

  1. Agris P. F., Fujiwara F. G., Schmidt C. F., Loeppky R. N. Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies. Nucleic Acids Res. 1975 Sep;2(9):1503–1512. doi: 10.1093/nar/2.9.1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agris P. F., Koh H., Söll D. The effect of growth temperatures on the in vivo ribose methylation of Bacillus stearothermophilus transfer RNA. Arch Biochem Biophys. 1973 Jan;154(1):277–282. doi: 10.1016/0003-9861(73)90058-1. [DOI] [PubMed] [Google Scholar]
  3. Beck C. F., Ingraham J. L., Neuhard J., Thomassen E. Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. J Bacteriol. 1972 Apr;110(1):219–228. doi: 10.1128/jb.110.1.219-228.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davis G. E., Gehrke C. W., Kuo K. C., Agris P. F. Major and modified nucleosides in tRNA hydrolysates by high-performance liquid chromatography. J Chromatogr. 1979 May 21;173(2):281–298. doi: 10.1016/s0021-9673(00)92297-0. [DOI] [PubMed] [Google Scholar]
  5. Eakin R. T., Morgan L. O., Gregg C. T., Matwiyoff N. A. Carbon-13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled 13C substrate. FEBS Lett. 1972 Dec 15;28(3):259–264. doi: 10.1016/0014-5793(72)80726-9. [DOI] [PubMed] [Google Scholar]
  6. Hamill W. D., Jr, Grant D. M., Horton W. J., Lundquist R., Dickman S. Letter: Magnetic resonance spectroscopy on carbon-13 labeled uracil in transfer ribonucleic acid. J Am Chem Soc. 1976 Mar 3;98(5):1276–1273. doi: 10.1021/ja00421a047. [DOI] [PubMed] [Google Scholar]
  7. Harris C. L., Titchener E. B., Cline A. L. Sulfur-deficient transfer ribonucleic acid in a cysteine-requiring, "relaxed" mutant of Escherichia coli. J Bacteriol. 1969 Dec;100(3):1322–1327. doi: 10.1128/jb.100.3.1322-1327.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hill D. L., Bennett L. L., Jr Purification and properties of 5-phosphoribosyl pyrophosphate amidotransferase from adenocarcinoma 755 cells. Biochemistry. 1969 Jan;8(1):122–130. doi: 10.1021/bi00829a017. [DOI] [PubMed] [Google Scholar]
  9. Horowitz J., Ofengand J., Daniel W. E., Jr, Cohn M. 19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli. J Biol Chem. 1977 Jun 25;252(12):4418–4420. [PubMed] [Google Scholar]
  10. Komoroski R. A., Allerhand A. Natural-abundance carbon-13 Fourier-transform nuclear magnetic resonance spectra and spin lattice relaxation times of unfractionated yeast transfer-FNA. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1804–1808. doi: 10.1073/pnas.69.7.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Komoroski R. A., Allerhand A. Observation of resonances from some minor bases in the natural-abundance carbon-13 nuclear magnetic resonance spectrum of unfractionated yeast transfer ribonucleic acid. Evidence for fast internal motion of the dihydrouracil rings. Biochemistry. 1974 Jan 15;13(2):369–372. doi: 10.1021/bi00699a023. [DOI] [PubMed] [Google Scholar]
  12. Murray A. W., Elliott D. C., Atkinson M. R. Nucleotide biosynthesis from preformed purines in mammalian cells: regulatory mechanisms and biological significance. Prog Nucleic Acid Res Mol Biol. 1970;10:87–119. doi: 10.1016/s0079-6603(08)60562-0. [DOI] [PubMed] [Google Scholar]
  13. NIERLICH D. P., MAGASANIK B. REGULATION OF PURINE RIBONUCLEOTIDE SYNTHESIS BY END PRODUCT INHIBITION. THE EFFECT OF ADENINE AND GUANINE RIBONUCLEOTIDES ON THE 5'-PHOSPHORIBOSYL-PYROPHOSPHATE AMIDOTRANSFERASE OF AEROBACTER AEROGENES. J Biol Chem. 1965 Jan;240:358–365. [PubMed] [Google Scholar]
  14. Ramberg E. S., Ishaq M., Rulf S., Moeller B., Horowitz J. Inhibition of transfer RNA function by replacement of uridine and uridine-derived nucleosides with 5-fluorouridine. Biochemistry. 1978 Sep 19;17(19):3978–3985. doi: 10.1021/bi00612a016. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Tompson J. G., Hayashi F., Paukstelis J. V., Loeppky R. N., Agris P. F. Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched transfer ribonucleic acid. Biochemistry. 1979 May 15;18(10):2079–2085. doi: 10.1021/bi00577a037. [DOI] [PubMed] [Google Scholar]
  17. Ward D. C., Reich E., Stryer L. Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives. J Biol Chem. 1969 Mar 10;244(5):1228–1237. [PubMed] [Google Scholar]
  18. Yang C. H., Söll D. Studies of transfer RNA tertiary structure of singlet-singlet energy transfer. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2838–2842. doi: 10.1073/pnas.71.7.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES