Granneman Sander, Bernstein Kara A, Bleichert Franziska, Baserga Susan J
Molecular Biophysics & Biochemistry Department, Yale University School of Medicine, 333 Cedar St., SHM C-114, New Haven, CT 06520-8024, USA.
Mol Cell Biol. 2006 Feb;26(4):1183-94. doi: 10.1128/MCB.26.4.1183-1194.2006.
The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.
前体核糖体RNA加工所需的17种假定的RNA解旋酶预计通过驱动前核糖体内部的结构重排，在核糖体生物合成中发挥关键作用。为了更好地理解这些蛋白质的功能，我们在参与18S rRNA合成的五种假定的RNA解旋酶中产生了一系列突变，并分析了它们对细胞生长和前体核糖体RNA加工的影响。我们的结果确定了保守基序内功能上重要的残基，并证明预测的ATP结合水解基序中的致死突变在野生型背景中过表达时，在体内通常会赋予显性负性表型。我们表明，显性负性突变体延迟了35S前体核糖体RNA的加工，并导致通常具有低稳态水平的前体核糖体RNA种类的积累。我们的综合结果表明，并非所有保守结构域在每种蛋白质中的功能都相同，这表明RNA解旋酶在核糖体生物合成中可能具有不同的生化特性和多种作用。
