Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.
Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.
复制标题
大肠杆菌 16S 核糖体 RNA 内核糖体蛋白 S8 结合位点的定点诱变。
DOI：
10.1093/nar/14.14.5761
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发表时间：
1986
影响因子：
14.9
通讯作者：
Zimmermann,RA
中科院分区：
文献类型：
--
作者：
Gregory,RJ;Zimmermann,RA
在大肠杆菌16 SrRNA中核糖体蛋白S8的结合位点上引入了12个特异性的改变。首先将合适的rDNA片段克隆到噬菌体M13载体中，经亚硫酸氢盐和阿糖胞苷体外诱变，然后将诱变后的序列插入质粒pN 01301的rrnB操纵子中，用突变质粒转化E.大肠杆菌受体。测定含有突变质粒的细胞的生长速率，并与含有野生型质粒的细胞的生长速率进行比较。只有那些发生在高度保守位置的突变，或预期会破坏结合位点的二级结构的突变，才明显增加了倍增时间。生长速率最显著的变化是由改变S8结合位点内一个小内环的突变引起的。这种结构在原核生物16 S rRNA中是遗传上保守的，并且可能在S8- 16 S rRNA识别和相互作用中起直接作用。
Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 inEscherichla coli16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesisin vitro.Subsequently, the mutagenlzed sequences were placed within the rrnB operon of plasmid pN01301 and the mutant plasmids were used to transformE. colirecipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetlcally conserved in prokaryotic 16S rRNAs and may play a direct role In S8-16S rRNA recognition and interaction.