Interaction analysis between tmRNA and SmpB from Thermus thermophilus.
Interaction analysis between tmRNA and SmpB from Thermus thermophilus.
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DOI：
10.1093/jb/mvi180
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发表时间：
2005-12
影响因子：
2.7
通讯作者：
N. Nameki;T. Someya;S. Okano;Reiko Suemasa;M. Kimoto;K. Hanawa-Suetsugu;T. Terada;M. Shirouzu;I. Hirao;H. Takaku;H. Himeno;A. Muto;S. Kuramitsu;S. Yokoyama;G. Kawai
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文献类型：
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作者：
N. Nameki;T. Someya;S. Okano;Reiko Suemasa;M. Kimoto;K. Hanawa-Suetsugu;T. Terada;M. Shirouzu;I. Hirao;H. Takaku;H. Himeno;A. Muto;S. Kuramitsu;S. Yokoyama;G. Kawai
小分子蛋白B，Smp B，是一种转录所必需的tRNA特异性结合蛋白。我们研究了从嗜热栖热菌的SmpB和tmRNA之间的相互作用，使用生化和NMR方法。使用全长tmRNA的化学足迹分析表明，在SmpB结合时受到保护的位点仅位于tmRNA的tRNA样结构域（tRNA）中。为了阐明SmpB的结合位点，我们构建了几个片段，来自于DNA。光学生物传感器相互作用分析和突变研究的熔解曲线分析表明，SmpB有效地结合到只有一个30-nt的片段，形成一个茎和环，与5'和3'延伸组成的D-环和可变环类似物。延伸区中的保守序列16 UCGA和319 GAC负责SmpB结合。这些结果与风产液囊菌（Aquifex aeolicus）中的SmpB和BMPB的共晶结构所显示的结果一致。此外，使用30-nt片段和（15）N-标记的SmpB的NMR化学位移图分析揭示了特征性的RNA结合模式。β 2周围的氢键模式发生变化，其中β 2中的Gly作为铰链，显示出最大的化学位移变化。似乎SmpB经历了结构变化，表明在结合到特定区域时的诱导拟合。
Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.