Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing.
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.
T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
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D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D005782:Gels; D035001:Host Factor 1 Protein / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012329:RNA, Bacterial / Q000378:metabolism*
D029968:Escherichia coli Proteins; D005782:Gels; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D050505:Mutant Proteins; D012329:RNA, Bacterial