Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA .1. Defining the minimal RNA site
Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA .1. Defining the minimal RNA site
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DOI：
10.1006/jmbi.1996.0481
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发表时间：
1996-08-30
影响因子：
5.6
通讯作者：
Williamson, JR
中科院分区：
文献类型：
--
作者：
Batey, RT;Williamson, JR
使用源自 16 S rRNA 中心结构域的合成 RNA 寡核苷酸分析嗜热脂肪芽孢杆菌核糖体蛋白 S15 (BS15) 的核糖体 RNA 结合位点。天然凝胶电泳迁移率变动分析表明，BS15 可以与含有 16 S rRNA 的核苷酸 585 至 756（螺旋 20 至 23）的 RNA 寡核苷酸特异性相互作用，表观解离常数为 35 nM。使用竞争结合测定法测试了一系列包含 BS15 特异性结合位点的 rRNA 片段缺失突变体结合蛋白质的能力。 BS15-rRNA相互作用的主要决定因素是螺旋20、21和22之间的三向连接，而螺旋23（16S rRNA的核苷酸673至733）对于高亲和力结合是可有可无的。螺旋 22 在一个内部环中包含 BS15 结合决定簇，该内部环包含两个系统发育上保守的嘌呤-嘌呤碱基对。相反，仅需要螺旋20和21的小片段来维持连接的完整性。 BS15 和各种最小 rRNA 结合位点之间的双分子复合物的解离和结合速率的动力学测量表明，这种相互作用的基本特性并未因删除而改变。最小结合位点是 61 个核苷酸的 RNA，它是野生型 BS15-16 S rRNA 相互作用的良好模型。 (C) 1996 学术出版社有限公司
The ribosomal RNA binding site of Bacillus stearothermophilus ribosomal protein S15 (BS15) was analyzed using synthetic RNA oligonucleotides derived from the 16 S rRNA central domain. Native gel electrophoresis mobility shift assays demonstrate that BS15 can specifically interact with an RNA oligonucleotide containing nucleotides 585 to 756 (helices 20 to 23) of 16 S rRNA with an apparent dissociation constant of 35 nM. A series of deletion mutants of the rRNA fragment that contains the BS15 specific binding site was tested for their capacity to bind protein using a competition binding assay. The major determinant of the BS15-rRNA interaction is a three-way junction between helices 20, 21, and 22, while helix 23 (nucleotides 673 to 733 of 16 S rRNA) was dispensable for high affinity binding. Helix 22 contains BS15 binding determinants in an internal loop containing two phylogenetically conserved purine-purine base-pairs. In contrast, only small segments of helices 20 and 21 are required to maintain the integrity of the junction. Kinetic measurements of the dissociation and association rate of the bimolecular complex between BS15 and various minimal rRNA binding sites demonstrate that the basic properties of this interaction were not altered as a result of the deletions. The minimal binding site is a 61 nucleotide RNA that is a good model for the wild-type BS15-16 S rRNA interaction. (C) 1996 Academic Press Limited