Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies.
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.
D002247:Carbon Isotopes; D004926:Escherichia coli / Q000032:analysis; D006151:Guanosine / Q000031:analogs & derivatives / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial; D012343:RNA, Transfer; D012266:Ribose; D013936:Thymidine / Q000031:analogs & derivatives / Q000032:analysis
D002247:Carbon Isotopes; D012329:RNA, Bacterial; D006151:Guanosine; D012266:Ribose; D012343:RNA, Transfer; D013936:Thymidine