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Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Nature Structural & Molecular Biology volume 11, pages 1206–1214 (2004)Cite this article

Abstract

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in post-transcriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.

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Figure 1: Sequence alignments and structure of Hfq.
Figure 2: In vitro analysis of RNA binding to mutant Hfq proteins.
Figure 3: Native gel analysis showing the effect of A27 addition to the DsrA–(Hfq6)2 complex.
Figure 4: Assay for in vivo accumulation and rpoS activation.
Figure 5: Lifetime analysis of DsrA in vivo.

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Acknowledgements

This work is supported by grants from the US National Institutes of Health (GM065430 to A.L.F., GM056448 to D.S., and T32-GM07757 to Indiana University/P.J.M.). A.L.F. is a Cottrell Scholar of Research Corporation. The Typhoon 9210 imaging system was purchased with a grant from the US National Science Foundation (DBI-0244815). The authors thank T. Stone (Indiana University) for technical support in the Physical Biochemistry Instrumentation Facility, and J. Fitzgerald, A. Kerzmann, N. Anderson and S. Kamel (Indiana University) for assistance in the cloning and sequencing of the constructs.

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  1. Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, 47405, Indiana, USA

    Peter J Mikulecky, Jennifer C Takach & Andrew L Feig

  2. Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, 43614-5806, Ohio, USA

    Meenakshi K Kaw, Cristin C Brescia & Darren D Sledjeski

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  1. Peter J Mikulecky
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  2. Meenakshi K Kaw
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Correspondence to Andrew L Feig.

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Supplementary information

Supplementary Figure 1

Isothermal titration calorimetry of DsrA titrated into wild-type Hfq. (PDF 62 kb)

Supplementary Table 1

Summary of thermodynamic data from ITC analysis. (PDF 8 kb)

Supplementary Table 2

Primers used for cloning and QRT-PCR. (PDF 12 kb)

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Mikulecky, P., Kaw, M., Brescia, C. et al. Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol 11, 1206–1214 (2004). https://doi.org/10.1038/nsmb858

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