Konikkat Salini, Biedka Stephanie, Woolford John L
Department of Biological Sciences, Carnegie Mellon University, 616 Mellon Institute, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
Nucleic Acids Res. 2017 May 5;45(8):4853-4865. doi: 10.1093/nar/gkw1361.
A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40-60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161-200 and erb1Δ201-245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.
我们对核糖体组装的理解存在一个重大差距，即了解约200种组装因子中每一种的确切功能。这些因子参与的亚基组装步骤在很大程度上是通过从细胞中耗尽每种蛋白质来研究的。组装因子Erb1的缺失会阻止其他七种相互依赖的组装因子与60S前体亚基的稳定组装，导致早期前核糖体的周转，此时ITS1间隔区还无法从27SA3前体rRNA中去除。为了研究Erb1更具体的功能，我们构建了八个内部缺失突变体，每个缺失40 - 60个氨基酸残基，跨越Erb1的氨基末端一半区域。erb1Δ161 - 200和erb1Δ201 - 245缺失突变阻断的步骤比Erb1缺失所阻断的步骤更靠后，即启动ITS2间隔区去除的C2位点的切割。在这些erb1突变体中，另外两个重塑事件也无法发生：十二种不同的组装因子与25S rRNA的结构域V结合，包括肽基转移酶中心周围区域，以及核糖体蛋白与多肽出口通道周围的rRNA稳定结合。这表明这些功能中心构建的成功起始是决定间隔区去除的一个检查点。
