Formenoy L J, Cunningham P R, Nurse K, Pleij C W, Ofengand J
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Biochimie. 1994;76(12):1123-8. doi: 10.1016/0300-9084(94)90040-x.
An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 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 that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition.
利用为大肠杆菌核糖体16S rRNA位点特异性诱变开发的体外系统，在3'末端发夹结构中高度保守的U1512.G1523碱基对周围进行了五个突变。每个突变RNA都与30S蛋白质的完整混合物重构，以产生30S核糖体颗粒，测试ksgA甲基化酶形成m6(2)A1518和m6(2)A1519的能力。发夹环中A1518和A1519的二甲基化被突变抑制了20 - 80%。结果表明，茎中的G1523和C1524是环中A1518和A1519二甲基化的重要决定因素。要么酶识别区域延伸到那么远，要么茎中突变的影响以某种方式传递到环。保守的U.G碱基对本身似乎在ksgA甲基化酶识别中不发挥主要作用。
