A carbon-13 nuclear magnetic resonance study of the 3'-terminus of 16S nbosomal RNA of Escherichia col specifically labeled with carbon-13 in the methylgroups of the mjAm§A sequence

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-(13C-methyl)-L-methionine chloride ((13C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacterio- cin cloacin DF13, 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 (m6A) and 6-N-dimethyladenylyl- (31+5') -6-N-dimethyladenosine (m6Am6A). The data show that the two methylated adenines, which are part of a four member-ed hairpin loop show a strong tendency to be stacked in analogy to the di- nucleotide m26Am6A. (2). In previous work (3-7) we have studied the influence of the methylgroups on the function of the ribosome and on the structure of the 16S RNA. Recent UV melting experiments have been carried out on the RNA fragment that results from cleavage by the bacteriocin cloacin DF13 and comprises 49 nucleotides from the 3'-end of the 16S RNA (8) (cloacin-fragment), part of which forms the above mentioned hairpin. Comparison of the results obtained for the fragment obtained from wild type E. coli with those obtained for the fragment from a mutant strain of E. coli that specifically lacks the methylgroups on the two A residues shows that the methylgroups have a subtle but definite influence on the thermodynamic properties of the hairpin. It was found that the helix in the wild type fragment was somewhat less stable compared to the corresponding helix in the mutant fragment. This was