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首页> 外文期刊>Journal of Molecular Biology >Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing
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Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing

机译：六聚体到溶液中大肠杆菌Hfq的单体平衡及其对RNA退火的影响

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摘要

The bacterial Sm-like protein Hfq forms a ring-shaped homo-hexamer that is necessary for Hfq to bind nucleic acids and to act in small noncoding RNA regulation. Using semi-native gels and fluorescence anisotropy, we show that Hfq undergoes a cooperative conformational change from monomer to hexamer around 1 μM protein, which is comparable to the in vivo concentration of Hfq and above the dissociation constant of the Hfq hexamer from many RNA substrates. Above 2μM protein, Hfq hexamers associate in high-molecular-weight complexes. Mutations that impair RNA binding to the proximal face strongly destabilize the hexamer, while the mutation R16A near the outer rim prevents hexamer association. Stopped-flow fluorescence resonance energy transfer experiments showed that Hfq subunits interact within a few seconds, suggesting that Hfq monomers, hexamers and multi-hexamer complexes are in dynamic equilibrium. Finally, we show that Hfq is most active in RNA annealing when the hexamer is present. These results suggest that RNA binding is coupled to hexamer assembly and that the biochemical activity of Hfq reflects the equilibrium between different quaternary structures.
机译：细菌Sm样蛋白Hfq形成环状同六聚体，这是Hfq结合核酸并在小的非编码RNA调控中起作用所必需的。使用半天然凝胶和荧光各向异性，我们显示Hfq在1μM蛋白质附近经历了从单体到六聚体的协同构象变化，这与Hfq的体内浓度相当，并且高于Hfq六聚体从许多RNA底物中的解离常数。蛋白质高于2μM时，Hfq六聚体会与高分子量复合物缔合。削弱RNA与近端结合的突变会严重破坏六聚体的稳定性，而靠近外缘的R16A突变会阻止六聚体缔合。停止流荧光共振能量转移实验表明，Hfq亚基在数秒内相互作用，这表明Hfq单体，六聚体和多六聚体络合物处于动态平衡。最后，我们表明当六聚体存在时，Hfq在RNA退火中最活跃。这些结果表明，RNA结合与六聚体组装偶联，并且Hfq的生化活性反映了不同四级结构之间的平衡。

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