title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.
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-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.
authors.affiliation: Department of Biochemistry, Duke University, Durham, NC 27710, USA and Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Kirsten E
authors.initials: KE
authors.lastname: Robinson
authors.forename: Jillian
authors.initials: J
authors.lastname: Orans
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Kovach
authors.forename: Todd M
authors.initials: TM
authors.lastname: Link
authors.forename: Richard G
authors.initials: RG
authors.lastname: Brennan
mesh_terms: D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D035001:Host Factor 1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008089:Listeria monocytogenes; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013211:Staphylococcus aureus; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D020121:5' Untranslated Regions; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012313:RNA; D014364:Tryptophan