Lanier Kathryn A, Roy Poorna, Schneider Dana M, Williams Loren Dean
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia.
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia.
Biophys J. 2017 Jul 25;113(2):268-276. doi: 10.1016/j.bpj.2017.04.007. Epub 2017 May 12.
We have proposed that the ancient ribosome increased in size during early evolution by addition of small folding-competent RNAs. In this Accretion Model, small RNAs and peptides were subsumed onto subunit surfaces, gradually encasing and freezing previously acquired components. The model predicts that appropriate rRNA fragments have inherited local autonomy of folding and local autonomy of assembly with ribosomal proteins (rProteins), and that the rProtein and rRNA are co-chaperones. To test these predictions, we investigate the rRNA interactions of rProtein uL23 and its tail, uL23, which is a β-hairpin that penetrates deep into the core of the large ribosomal subunit. In the assembled ribosome, uL23 associates with Domain III of the rRNA and a subdomain called "DIII". Here using band shift assays, fluorescence Job plots, and yeast three-hybrid assays, we investigate the interactions of rProtein uL23 and its tail with Domain III and with DIII rRNA. We observe rRNA-uL23 complexes in the absence of Mg ions and rRNA-uL23 (n > 1) complexes in the presence of Mg ions. By contrast, the intact uL23 rProtein binds in slightly anticooperative complexes of various stoichiometries. The globular and tail regions of rProtein uL23 are distinctive in their folding behaviors and the ion dependences of their association with rRNA. For the globular region of the rProtein, folding is independent of rRNA, and rRNA association is predominantly by nonelectrostatic mechanisms. For the tail region of the protein, folding requires rRNA, and association is predominantly by electrostatic mechanisms. We believe these protein capabilities could have roots in ancient evolution and could be mechanistically important in co-chaperoning the assembly of the ribosome.
我们提出，在早期进化过程中，古老的核糖体通过添加具有折叠能力的小RNA而增大了尺寸。在这个“ accretion模型”中，小RNA和肽被纳入亚基表面，逐渐包裹并固定先前获得的成分。该模型预测，合适的rRNA片段继承了与核糖体蛋白（rProtein）折叠的局部自主性和组装的局部自主性，并且rProtein和rRNA是共同伴侣。为了验证这些预测，我们研究了rProtein uL23及其尾巴uL23的rRNA相互作用，uL23是一个深入到大核糖体亚基核心的β-发夹结构。在组装好的核糖体中，uL23与rRNA的结构域III和一个称为“ DIII”的亚结构域结合。在这里，我们使用带移分析、荧光Job图和酵母三杂交分析，研究rProtein uL23及其尾巴与结构域III和DIII rRNA的相互作用。我们在没有镁离子的情况下观察到rRNA-uL23复合物，在有镁离子的情况下观察到rRNA-uL23（n>1）复合物。相比之下，完整的uL23 rProtein以各种化学计量比的轻微反协同复合物形式结合。rProtein uL23的球状区域和尾巴区域在其折叠行为以及与rRNA结合的离子依赖性方面是不同的。对于rProtein的球状区域，折叠独立于rRNA，并且rRNA结合主要通过非静电机制。对于蛋白质的尾巴区域，折叠需要rRNA，并且结合主要通过静电机制。我们认为这些蛋白质能力可能起源于古老的进化，并且在共同陪伴核糖体组装方面可能具有重要的机制意义。
