Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing

Subrata Panja, Andrew Santiago-Frangos, Daniel J. Schu, Susan Gottesman, Sarah A. Woodson

Research output: Contribution to journalArticlepeer-review

Abstract

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.

Original languageEnglish (US)
Pages (from-to)3491-3500
Number of pages10
JournalJournal of molecular biology
Volume427
Issue number22
DOIs
StatePublished - May 25 2015

Keywords

  • Hfq
  • RNA chaperone
  • RNA protein interactions
  • molecular beacon
  • small non-coding RNA

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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