A Carbon-13 Nuclear Magnetic Resonance Study of the 3'-Terminus of 16S Ribosomal RNA of Escherichia coli Specifically Labeled With Carbon-13 in the Methylgroups of the Di(Dimethyladenosine)

30S ribosomes were isolated from a kasugamycin resistant mutant of E. coli that lacks methylgroups on two adjacent adenines in 16S ribosomal RNA. These ribosomes were methylated in vitro with a purified methylating enzyme and 5-S-adenosyl-( super(13)C-methyl)-L-methionine chloride (( super(13)C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacteriocin cloacin DF sub(13), the 49 nucleotide fragment from the 3'-end of the RNA was isolated. The carbon-13 nuclear magnetic resonance spectra of the fragment at various temperatures were compared with those of 6-N-dimethyladenosine and 6-N-dimethyladenylyl-(3' arrow right 5')-6-N-dimethyladenosine. The data show that the two methylated adenines, which are part of a four membered hairpin loop show a strong tendency to be stacked.