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Nucleic Acids Research, 1975, Vol. 2, No. 9 1503-1512
© 1975

Articles

Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies

Paul F. Agris, F.Genichi Fujiwara, Charles F. Schmidt and Richard N. Loeppky

Division of Biological Sciences and Department of Chemistry, University of Missouri Columbia, MO 65201, USA

Received June 4, 1975. The enrichment of tRNA at specific sites with carbon-13 has been accomplished in vivo using a mutant of Escherichia coli. A relaxed strain of E. coli auxotrophic for methionine was grown in a specifically defined medium supplemented with either [14C] or [13C]-methyl labeled methionine. Cells were collected at the end of the log-phase of growth and tRNA was extracted. Analysis of the radioactivity of the [14C]-labeled tRNA established an incorporation ratio of three labeled carbons per tRNA molecule. Incorporation of the [14C]-label in vivo was confined to the methylation of nucleotides as determined by thin layer chromatography of nucleotides resulting from a ribonuclease digestion of [14C]-labeled tRNA. The carbon-13 NMR spectrum of [13C]- enriched tRNA indicated a similar degree of incorporation into the methylated nucleotides by the substantial enhancement of [13C]-methyl NMR signals only. Assignment of signals has been made for the methyl groups of ribothymidine and N -methylguanosine in E. coli tRNA.


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