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. 1975 Sep;2(9):1503–1512. doi: 10.1093/nar/2.9.1503

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

P F Agris, F G Fujiwara, C F Schmidt, R N Loeppky
PMCID: PMC343518  PMID: 1101225

Abstract

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 N7-methylguanosine in E. coli tRNA.

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Selected References

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

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