T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
J Mol Biol. 2012 Apr 13;417(5):406-12. doi: 10.1016/j.jmb.2012.02.009. Epub 2012 Feb 10.
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 2 μM 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.
细菌 Sm 样蛋白 Hfq 形成一个环形同型六聚体，这对于 Hfq 结合核酸并在小非编码 RNA 调节中发挥作用是必要的。使用半天然凝胶和荧光各向异性，我们表明 Hfq 在 1μM 左右的蛋白浓度下经历从单体到六聚体的协同构象变化，这与体内 Hfq 浓度相当，高于许多 RNA 底物的 Hfq 六聚体从其解离常数。在 2μM 以上的蛋白浓度下，Hfq 六聚体以高分子量复合物的形式结合。破坏 RNA 与近端面结合的突变强烈破坏六聚体，而外边缘附近的 R16A 突变则阻止六聚体的结合。停流荧光共振能量转移实验表明，Hfq 亚基在几秒钟内相互作用，表明 Hfq 单体、六聚体和多六聚体复合物处于动态平衡中。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。
