Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics
Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics
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DOI：
10.1073/pnas.0508444102
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发表时间：
2005-11-15
影响因子：
11.1
通讯作者：
Mankin, AS
中科院分区：
文献类型：
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作者：
Yassin, A;Fredrick, K;Mankin, AS
许多临床上有用的抗生素通过抑制核糖体功能来干扰细菌病原体中的蛋白质合成。已知药物的作用位点数量有限，主要由核糖体RNA（rRNA）组成，并且与核糖体的功能关键中心一致。这些位点内的核苷酸改变通常是有害的。为了确定核糖体中抗生素作用的功能位点和潜在位点，我们制备了rRNA基因的随机突变体文库，并选择了干扰细胞生长的16S rRNA中的显性突变。53个16S rRNA位点被鉴定出其突变抑制蛋白质合成。根据表型的严重程度对突变进行排序，并且在专门的核糖体系统中验证了几种突变对翻译的有害影响。突变体的多核糖体概况的分析表明，大多数的突变直接干扰核糖体的功能，而一小部分的突变影响装配的小核糖体亚基。12个已鉴定的突变定位于已知抗生素靶向的位点，证实了有害突变可用于鉴定抗生素靶点。大约一半的突变与核糖体中已知的功能位点相吻合，而其余的突变影响核糖体位点，功能意义不太明确。在其他不起眼的核糖体位点的有害突变的四个集群进行了鉴定，表明其功能的重要性和潜在的抗生素的目标。
Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.