METHYLATION OF MINIMALIST 23S RIBOSOMAL-RNA SEQUENCES IN-VITRO BY ERMSF (TLRA) N-METHYLTRANSFERASE

ermSF (synonym tlrA) from Streptomyces fradiae NRRL 2702 confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) superfamily of antibiotics. ErmSF specifically methylates Bacillus subtilis 23S rRNA in vitro at A2085 (B. subtilis coordinate, which is equivalent to the Escherichia coli coordinate A2058). In the present studies, partial B. subtilis 23S rRNA sequences containing portions of the peptidyltransferase circle which include A2085 were constructed in order to identify structural requirements needed for RNA to function as substrate of ErmSF, A model methylase substrate based on the 41-nucelotide construct DK111, ggCCUAUCCGUCGCGGGUUCGCCCGCGACAGGACGGA*AAGA, had methyl-acceptor activity. This sequence contains 235 rRNA stem 73 [Stade, K,, et al, (1994) Nucleic Acids Res. 22, 1394-1399] underlined, flanking a tetraloop-like (UUCG), and the unpaired sequence AAAGA, at the 3' end containing A2085 (A*). A set of systematic alterations introduced into the sequence suggested that the four unpaired nucleotides in stem 73 are necessary for methyl-acceptor activity, whereas inversion of 11 out of 13 paired bases in stem 73 conferred no significant reduction in methyl-acceptor activity.