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Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly

机译：中央枢纽控制30S核糖体装配中的蛋白质RNA动力学。

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摘要

Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Forster or fluorescence resonance energy transfer (smFRET) was used to study the Mg2+- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function. (C) 2016 Elsevier Ltd. All rights reserved.
机译：核糖体蛋白（r蛋白）和核糖体RNA（rRNA）之间的相互作用促进了功能性核糖体的形成。 S15是中央结构域一级结合蛋白，已显示可触发16S rRNA构象级联变化，形成中央结构域的功能结构。先前的体外生物化学和结构研究表明，S15结合螺旋20、21和22的三向连接，包括核苷酸652-654和752-754。除653外，所有连接核苷酸在细菌中都是高度保守的。为了鉴定连接内功能上重要的基序，我们对核苷酸652-654和752-754进行了饱和诱变，并选择和分析了功能突变体。仅分离了体内具有大于10％核糖体功能的突变体。 S15的过表达补充了每个部分活性突变体的连接环中的突变，尽管产生无活性核糖体的突变并未被S15的过表达所补充。使用单分子Forster或荧光共振能量转移（smFRET）来研究Mg2 +和S15诱导的所选连接突变体的构象动力学。在存在和不存在S15的情况下，将这些突变体与野生型的结构动力学进行比较，发现在中央连接处的特定序列和结构基序对核糖体功能很重要。 （C）2016 Elsevier Ltd.保留所有权利。

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