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Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.

Publication ,  Journal Article
Robinson, KE; Orans, J; Kovach, AR; Link, TM; Brennan, RG
Published in: Nucleic Acids Res
February 2014

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5'-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

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Published In

Nucleic Acids Res

DOI

EISSN

1362-4962

Publication Date

February 2014

Volume

42

Issue

4

Start / End Page

2736 / 2749

Location

England

Related Subject Headings

  • Tryptophan
  • Staphylococcus aureus
  • RNA
  • Protein Binding
  • Mutation
  • Models, Molecular
  • Listeria monocytogenes
  • Host Factor 1 Protein
  • Fluorescence
  • Escherichia coli Proteins
 

Citation

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Robinson, K. E., Orans, J., Kovach, A. R., Link, T. M., & Brennan, R. G. (2014). Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching. Nucleic Acids Res, 42(4), 2736–2749. https://doi.org/10.1093/nar/gkt1171
Robinson, Kirsten E., Jillian Orans, Alexander R. Kovach, Todd M. Link, and Richard G. Brennan. “Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.Nucleic Acids Res 42, no. 4 (February 2014): 2736–49. https://doi.org/10.1093/nar/gkt1171.
Robinson KE, Orans J, Kovach AR, Link TM, Brennan RG. Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching. Nucleic Acids Res. 2014 Feb;42(4):2736–49.
Robinson, Kirsten E., et al. “Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.Nucleic Acids Res, vol. 42, no. 4, Feb. 2014, pp. 2736–49. Pubmed, doi:10.1093/nar/gkt1171.
Robinson KE, Orans J, Kovach AR, Link TM, Brennan RG. Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching. Nucleic Acids Res. 2014 Feb;42(4):2736–2749.
Journal cover image

Published In

Nucleic Acids Res

DOI

EISSN

1362-4962

Publication Date

February 2014

Volume

42

Issue

4

Start / End Page

2736 / 2749

Location

England

Related Subject Headings

  • Tryptophan
  • Staphylococcus aureus
  • RNA
  • Protein Binding
  • Mutation
  • Models, Molecular
  • Listeria monocytogenes
  • Host Factor 1 Protein
  • Fluorescence
  • Escherichia coli Proteins