BipA is a conserved translational GTPase of bacteria recently implicated in ribosome biogenesis. Here we show that Escherichia coli ΔbipA cells grown at suboptimal temperature accumulate immature large subunit particles missing several proteins. These include L17 and L17-dependent binders, suggesting that structural block 3 of the subunit folds late in the assembly process. Parallel analysis of the control strain revealed accumulation of nearly identical intermediates, albeit at lower levels, suggesting qualitatively similar routes of assembly. This came as a surprise, because earlier analogous studies of wild-type E. coli showed early binding of L17. Further investigation showed that the main path of 50S assembly differs depending on conditions of growth. Either supplementation of the media with lysine and arginine or suboptimal temperature appears to delay block 3 folding, demonstrating the flexible nature of the assembly process. We also show that the variant BipA-H78A fails to rescue phenotypes of the ΔbipA strain, indicating a critical role for GTP hydrolysis in BipA function. In fact, BipA-H78A confers a dominant negative phenotype in wild-type cells. Controlled production of BipA-H78A causes accumulation of 70S monosomes at the expense of polysomes, suggesting that the growth defect stems from a shutdown of translation.

BipA 是最近与核糖体生物发生有关的细菌的保守翻译 GTPase。在这里，我们展示了在次优温度下生长的大肠杆菌 ΔbipA细胞积累了缺失几种蛋白质的未成熟大亚基颗粒。这些包括 L17 和 L17 依赖性结合剂，表明亚基的结构块 3 在组装过程后期折叠。对照菌株的平行分析揭示了几乎相同的中间体的积累，尽管水平较低，表明组装路线在质量上相似。这令人惊讶，因为早期对野生型大肠杆菌的类似研究显示出 L17 的早期结合。进一步研究表明，50S 组装的主要途径因生长条件而异。用赖氨酸和精氨酸补充培养基或次优温度似乎会延迟块 3 折叠，证明组装过程的灵活性。我们还表明变体 BipA-H78A 未能挽救ΔbipA菌株的表型，表明 GTP 水解在 BipA 功能中起关键作用。事实上，BipA-H78A 在野生型细胞中赋予显性阴性表型。BipA-H78A 的受控生产以多核糖体为代价导致 70S 单体的积累，这表明生长缺陷源于翻译的关闭。