Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Robinson, Kirsten E.
Orans, Jillian
Kovach, Alexander R.
Link, Todd M.
Brennan, Richard G.

¹Department of Biochemistry, Duke University, Durham, NC 27710, USA and ²Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA

^*To whom correspondence should be addressed. Tel: +1 919 684 9471; Fax: +1 919 684 8885; Email: [email protected]

Received 3 September 2013; revised 29 October 2013; accepted 30 October 2013

Nucleic Acids Research 42(4):p 2736-2749, February 2014. | DOI: 10.1093/nar/gkt1171

ABSTRACT

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-negativeEscherichia coli(Ec) Hfq and the distal face binding of (AA)₃A, (AU)₃A and (AC)₃A to Gram-positiveStaphylococcus 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-positiveListeria 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 ofhfqmRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.