A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit.
A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit.
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DOI：
10.1093/nar/gkp711
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发表时间：
2009-10
影响因子：
14.9
通讯作者：
Uhlenbeck OC
中科院分区：
文献类型：
--
作者：
Sharpe Elles LM;Sykes MT;Williamson JR;Uhlenbeck OC
大肠杆菌DbpA是一种ATP依赖的RNA解旋酶，对23 S核糖体RNA的发夹92具有特异性，发夹92是肽基转移酶中心的重要组成部分。DbpA的R331A活性位点突变体在E.大肠杆菌中表达。来自过表达DbpA R331A的细胞的核糖体谱显示50S和30S亚基水平增加，70S核糖体水平降低。在低Mg 2+下运行的配置文件显示出更少的50S亚基，并积累了45S颗粒，该颗粒含有不完全加工和修饰不足的23S rRNA，以及在装配途径后期结合的几种核糖体蛋白的水平降低。与成熟的50S亚基不同，这些45S颗粒可以刺激DbpA的ATP酶活性，表明发夹92尚未被隔离在50S亚基内。过表达的无活性DbpA R331A突变体出现在后期阶段时，肽基转移酶中心的形成，表明可能的作用DbpA促进这种构象变化的装配块。
Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.
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