Melançon P, Lemieux C, Brakier-Gingras L
Département de Biochimie, Université de Montréal, Québec, Canada.
Nucleic Acids Res. 1988 Oct 25;16(20):9631-9. doi: 10.1093/nar/16.20.9631.
Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.
利用寡核苷酸定向诱变技术，在克隆于质粒pKK3535的大肠杆菌rrnB操纵子的16S核糖体RNA基因的第523位引入了一个从A到C的颠换。用突变体质粒转化的大肠杆菌细胞对链霉素具有抗性。从这些细胞中分离出的突变核糖体在聚（U）指导的实验中不会被链霉素刺激而误读信息。它们对其他促进错误的相关氨基糖苷类抗生素如新霉素、卡那霉素或庆大霉素刺激的误读也具有限制性，这些抗生素并不与链霉素结合位点竞争。16S rRNA发生突变的530环已定位在30S亚基的外表面，因此远离亚基界面处的链霉素结合位点。我们的结果支持这样的结论：16S rRNA第523位的突变不会干扰链霉素的结合，但会阻止药物诱导530环的构象变化，而这种构象变化是其错义编码效应的原因。由于这种效应主要是由翻译校对控制的扰动引起的，我们的结果也提供了证据表明16S rRNA的530环参与了这种准确性控制。
