Sharpe Elles Lisa M, Sykes Michael T, Williamson James R, Uhlenbeck Olke C
Department of Biochemistry, Northwestern University, Evanston, IL 60208, USA.
Nucleic Acids Res. 2009 Oct;37(19):6503-14. doi: 10.1093/nar/gkp711. Epub 2009 Sep 4.
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 Mg(2+) 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.
大肠杆菌DbpA是一种依赖ATP的RNA解旋酶，对23S核糖体RNA的发夹结构92具有特异性，该结构是肽基转移酶中心的重要组成部分。DbpA的R331A活性位点突变体在含有内源性DbpA的大肠杆菌中过表达时，会表现出显性的生长缓慢和冷敏感表型。过表达DbpA R331A的细胞的核糖体图谱显示，50S和30S亚基水平增加，70S核糖体水平降低。在低镁离子浓度下运行的图谱显示50S亚基较少，并积累了一种45S颗粒，该颗粒除了含有组装途径后期结合的几种核糖体蛋白水平降低外，还包含未完全加工和修饰不足的23S rRNA。与成熟的50S亚基不同，这些45S颗粒可以刺激DbpA的ATP酶活性，表明发夹结构92尚未被隔离在50S亚基内。无活性的DbpA R331A突变体的过表达似乎在肽基转移酶中心形成的后期阶段阻止组装，这表明DbpA在促进这种构象变化中可能发挥作用。
