Thompson J, Cundliffe E
Department of Biochemistry, University of Leicester, UK.
Biochimie. 1991 Jul-Aug;73(7-8):1131-5. doi: 10.1016/0300-9084(91)90156-u.
The antibiotic, thiostrepton, binds to 23S ribosomal RNA from E coli with a dissociation constant (KD) of 2.4 x 10(-7) M. The specificity of the interaction was established using 16S rRNA and modified or mutationally-altered 23S rRNA. Thus, no binding was detected with rRNA from the 30S subunit nor with rRNA modified in vitro by the thiostrepton resistance methylase. Mutant 23S rRNA, altered at residue 1067 in each of the 3 possible ways, showed reduced binding affinity for thiostrepton. The KD for the G mutation was 3.5 x 10(-6) M; for the C mutation, 2.4 x 10(-5) M; and for the U mutation, 4.8 x 10(-5) M. This reduction in drug binding is compatible with functional analyses; the C or U mutation results in ribosomal particles which are poorly inhibited by the drug compared with wild-type, whereas the G mutation results in an intermediate response to the drug in protein synthesis. The smallest 23S rRNA fragment used here that was capable of binding thiostrepton, in a nitrocellulose filter binding assay, comprised residues 1052-1112 and the dissociation constant was 3.0 x 10(-7) M, ie virtually indistinguishable from that with intact 23S RNA. However, the drug was incapable of binding to the 5'-moiety of this fragment (ie residues 1052-1084) or to an RNA transcript complementary to 1052-1112.
抗生素硫链丝菌素与来自大肠杆菌的23S核糖体RNA结合，解离常数（KD）为2.4×10（-7）M。利用16S rRNA以及经修饰或突变改变的23S rRNA确定了这种相互作用的特异性。因此，未检测到与30S亚基的rRNA结合，也未检测到经硫链丝菌素抗性甲基化酶体外修饰的rRNA结合。以三种可能方式之一在第1067位残基处发生改变的突变型23S rRNA对硫链丝菌素的结合亲和力降低。G突变的KD为3.5×10（-6）M；C突变的KD为2.4×10（-5）M；U突变的KD为4.8×10（-5）M。药物结合的这种降低与功能分析结果相符；C或U突变导致核糖体颗粒与野生型相比对药物的抑制作用较弱，而G突变导致在蛋白质合成中对药物产生中等反应。在硝酸纤维素滤膜结合试验中，此处使用的能够结合硫链丝菌素的最小23S rRNA片段包含第1052 - 1112位残基，解离常数为3.0×10（-7）M，即与完整的23S RNA几乎没有区别。然而，该药物无法与该片段的5'部分（即第1052 - 1084位残基）结合，也无法与与1052 - 1112互补的RNA转录本结合。
