Site-directed mutagenesis of ribosomal RNA. Construction and characterization of deletion mutants.
Site-directed mutagenesis of ribosomal RNA. Construction and characterization of deletion mutants.
复制标题
核糖体RNA的定点诱变。
DOI：
10.1016/0022-2836(82)90291-1
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发表时间：
1982
影响因子：
5.6
通讯作者：
A. E. Dahlberg
中科院分区：
文献类型：
--
作者：
R. Gourse;Michael J.R. Stark;A. E. Dahlberg
我们采用体外定点突变技术，对携带rnBrRNA操纵子的大肠杆菌质粒进行了遗传学研究，研究了核糖体RNA的结构和功能。通过从亲本质粒中选择的限制性位点进行有限的核酸外切酶消化，产生一系列的mu.构建了大肠杆菌菌株，每个菌株在对应于16 S rRNA的碱基614、704、1384和1504以及23 S rRNA的碱基365、607和1984的7个位置中的一个处具有缺失。我们选择了长度为1至30个碱基的小缺失质粒进行研究，以便将rRNA分子的不同区域与rRNA加工、其组装成核糖体亚基及其在核糖体中的功能相关联。通过限制性内切酶分析和DNA测序鉴定缺失的特征，并且突变的rRNA被证明在体内转录。除了正常的7个野生型rRNA顺反子外，携带改变的质粒的菌株的生长速度在许多情况下严重减慢。一些携带只有一个碱基缺失的质粒的菌株的倍增时间是携带未改变的质粒的菌株的两倍。在一个突变体的情况下，有一个自发的删除在第二个网站，部分抑制了23 S RNA.The方法的初始单碱基突变的影响，这里描述的含有rDNA突变的克隆的构建和繁殖提供了一种替代的方法rRNA的结构和功能的研究。Starket等人的随附论文描述了对改变的转录本的分析、它们的加工、组装成亚基以及通过允许它们与宿主编码的rRNA分开检测的方法掺入核糖体。
We have used a genetic approach to the study of ribosomal RNA structure and function by usingin vitro, site-directed mutagenesis techniques on a plasmid carrying therrnBrRNA operon ofEscherichia coli. By limited exonuclease digestion from chosen restriction sites in the parent plasmid, a series of mutantE. colistrains was constructed, each with a deletion at one of seven positions corresponding to bases 614, 704, 1384 and 1504 in 16 S rRNA and 365, 607 and 1984 in 23 S rRNA. We have selected for study plasmids containing small deletions of 1 to 30 bases in length in order to correlate distinct regions of the rRNA molecule with rRNA processing, its assembly into ribosomal subunits, and its function in ribosomes. The deletions were characterized by restriction enzyme analysis and DNA sequencing, and the mutant rRNAs were shown to be transcribedin vivo. The growth rates of strains that carry the altered plasmids, in addition to their normal complement of seven wild-type rRNA cistrons, are in many cases severely retarded. Some strains carrying plasmids with only a single base deletion have doubling times as much as twice that of the strain carrying the unaltered plasmid. In the case of one mutant, there is a spontaneous deletion at a second site that partially suppresses the effects of an initial single base mutation in 23 S RNA.The methodology described here for the construction and propagation of clones containing rDNA mutations provides an alternative approach to the study of rRNA structure and function. The accompanying paper by Starket al.describes the analysis of the altered transcripts, their processing, assembly into subunits, and incorporation into ribosomes by a method that allows their detection separately from the host-encoded rRNA.
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影响因子：
3.3
作者：
P. Howard-Flanders;L. Theriot
通讯作者：
P. Howard-Flanders;L. Theriot
影响因子：
56.9
作者：
NOLLER, HF;WOESE, CR
通讯作者：
WOESE, CR
影响因子：
5.6
作者：
Kingston,RE;Gutell,RR;Taylor,AR;Chamberlin,MJ
通讯作者：
Chamberlin,MJ
核糖体RNA的精细结构。
影响因子：
14.9
作者：
Gourse,RL;Gerbi,SA
通讯作者：
Gerbi,SA