METHYLATION OF THE CONSERVED A1518-A1519 IN ESCHERICHIA-COLI 16S-RIBOSOMAL RNA BY THE KSGA METHYLTRANSFERASE IS INFLUENCED BY METHYLATIONS AROUND THE SIMILARLY CONSERVED U1512-CENTER-DOT-G1523 BASE-PAIR IN THE3' TERMINAL HAIRPIN

An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations aro und the highly conserved U1512.G1523 base pair in the 3' terminal hair pin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested f or the ability of the ksgA methylase to form m(2)(6)A1518 and m(2)(6)A 1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibit ed 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends th at far or the effect of mutations in the stem are propagated in some m anner to the loop. The conserved U.G base pair does not of itself appe ar to play a major role in ksgA methylase recognition.