A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

中文翻译：

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四元驼背mRNA翻译抑制复合物的结构和动力学

果蝇发育过程中的一个关键调节过程是在后部对驼背 mRNA 翻译的局部抑制，这会产生控制前后体轴形成的驼背梯度。这种抑制是通过脑肿瘤 (Brat)、Pumilio (Pum) 和 Nanos 的协同作用实现的。每种蛋白质都是果蝇正常发育所必需的。已经在几个原子分辨率结构中单独阐明了蛋白质的 RNA 接触。然而，在 RNA 抑制过程中所有三种蛋白质的相互作用仍然是一个长期悬而未决的问题。在这里，我们通过将 NMR 光谱、SANS/SAXS、XL/MS 与 MD 模拟和 ITC 分析相结合，表征了 Brat、Pum 和 Nanos 的 RNA 结合结构域与驼背 mRNA 的四元复合物。包含 RNA 结合结构域的四元驼背 mRNA 抑制复合物是灵活的，未占据的核苷酸充当复合物的 Brat 和 Pum-Nanos 部分之间的灵活接头。此外，Pum-HD/Nanos-ZnF 复合物的存在对 Brat RNA 结合域的平衡 RNA 结合亲和力没有影响。这与之前的研究一致，表明Brat可以独立抑制mRNA，并且在整个胚胎中均匀分布。