Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Robinson, KE; Orans, J; Kovach, AR; Link, TM; Brennan, RG

Published Date

  • February 2014

Published In

Volume / Issue

  • 42 / 4

Start / End Page

  • 2736 - 2749

PubMed ID

  • 24288369

Pubmed Central ID

  • PMC3936774

Electronic International Standard Serial Number (EISSN)

  • 1362-4962

Digital Object Identifier (DOI)

  • 10.1093/nar/gkt1171


  • eng

Conference Location

  • England