Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

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 A(15) and U(6) RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)(3)A, (AU)(3)A and (AC)(3)A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)(3)G 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)(3)G 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.