Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly
Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly
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DOI：
10.1261/rna.067504.118
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发表时间：
2019-04-01
期刊：
影响因子：
4.5
通讯作者：
Bockelmann, Ulrich
中科院分区：
文献类型：
--
作者：
Geffroy, Laurent;Bizebard, Thierry;Bockelmann, Ulrich
体外重构研究表明，核糖体组装是高度协作的，并从一些核糖体 (r-) 蛋白与 rRNA 的结合开始。目前尚不清楚这些早期粘合剂如何发挥作用。着眼于大肠杆菌核糖体大亚基组装的初始阶段，我们制备了一个 79 个核苷酸长的 23S rRNA 区域，其中包含早期结合物 uL4 和 uL24 的结合位点。使用双光镊装置在 DNA/RNA 哑铃配置中测量力信号。在存在或不存在 r 蛋白（uL4、uL24 或两者）的情况下，rRNA 片段被拉伸直至展开。我们发现 r 蛋白 uL4 和 uL24 分别稳定 rRNA 片段，两者都充当分子夹。有趣的是，当两种蛋白质同时结合时，这种机械稳定性会增强。我们独立地观察到 uL4 和 uL24 与 rRNA 片段的协同结合。 r 蛋白结合的这两个方面都有助于有效稳定所研究的 rRNA 片段的 3D 结构。最后，我们考虑了我们的结果对大核糖体亚基组装的影响。
In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.