Interaction network of the ribosome assembly machinery from a eukaryotic thermophile
Interaction network of the ribosome assembly machinery from a eukaryotic thermophile
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DOI：
10.1002/pro.3085
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发表时间：
2017-02-01
期刊：
影响因子：
8
通讯作者：
Hurt, Ed
中科院分区：
文献类型：
--
作者：
Bassler, Jochen;Ahmed, Yasar Luqman;Hurt, Ed
真核细胞中的核糖体生物发生是一个高度动态和复杂的过程，与细胞增殖先天相关。核糖体的组装是由无数的生物发生因素驱动的，这些生物发生因素通过加工和折叠核糖体RNA并结合核糖体蛋白来形成核糖体前颗粒。生物化学方法允许从酿酒酵母中分离和表征核糖体前颗粒，从而绘制出从核仁到细胞质的生物发生中间体的时空图。在这里，我们克隆了几乎全部（大约180个）来自嗜热毛菌的核糖体生物发生因子，以便对它们的蛋白质-蛋白质相互作用网络进行深入分析，并探索这些耐热蛋白在结构研究中的适用性。首先，我们进行了系统筛选，测试了大约80个因素来确定结晶和结构。接下来，我们进行了酵母2-杂交分析，并测试了大约32,000个二元组合，其中发现了超过1000个嗜热核糖体组装因子之间的蛋白质-蛋白质接触。为了验证这些相互作用，我们进行了生化重构，重点研究了形成ctUTP-A和ctUTP-B模块的90S前核糖体因子与前60s亚基的brix结构域的相互作用网络。我们的工作为真核嗜热菌保守核糖体组装机制的生化重建和结构分析提供了丰富的资源。PDB代码：；
Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (approximate to 180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.PDB Code(s): ;