Escherichia coli Hfq Binds A18 and DsrA Domain II with Similar 2:1 Hfq6/RNA Stoichiometry Using Different Surface Sites

• Published in: Biochemistry (Easton) 2006-04, Vol.45 (15), p.4875-4887
• Main Authors: Sun, Xueguang, Wartell, Roger M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; Binding Sites; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Escherichia coli Proteins - pharmacology; Fluorescence Polarization; Gene Expression Regulation, Bacterial; Host Factor 1 Protein - chemistry; Host Factor 1 Protein - genetics; Host Factor 1 Protein - metabolism; Host Factor 1 Protein - pharmacology; Models, Molecular; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Protein Structure, Tertiary; RNA - chemistry; RNA - metabolism; RNA, Small Untranslated; RNA, Untranslated - chemistry; RNA, Untranslated - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Sequence Alignment; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0523613

Hfq is a RNA-binding protein in Escherichia coli that plays an essential role in post-transcriptional regulation of mRNAs by facilitating pairing of noncoding RNAs (ncRNAs) to mRNA target sites. Recent work has provided evidence that E. coli Hfq has two distinct RNA-binding surfaces. In this study, a comparative sequence−structure analysis of hfq genes in bacterial genomes was employed to identify conserved residues that may be involved in binding RNA. A covariance of residue properties at neighboring positions 12 and 39 and conserved surface residues with high propensities at binding sites of RNA-binding proteins suggested several sites for Hfq−RNA interactions. On the basis of these predictions, eight mutant Hfq proteins were produced and their interactions were examined with the 38 nucleotide (nt) domain II of DsrA ncRNA (DsrADII) and A18 by a gel-mobility shift assay, fluorescence anisotropy, and fluorescence quenching. Mutations on the proximal surface of Hfq had a small affect on Hfq binding to A18 (≤2-fold), while the mutations Y25A and K31A on the distal surface decreased affinity to A18 by 100-fold in solution. Mutations F39A and R16A on the proximal surface reduced affinity to DsrADII by 6−8-fold, while other mutations on the distal or proximal surfaces affected affinity to DsrADII by ≤2-fold using the gel-mobility shift assay. The F39A/L12F double mutation partially regained the affinity for DsrADII lost by the F39A mutation. The latter observation is consistent with the implied importance of an aromatic residue at position 12 or 39 suggested by the sequence covariance. Titration experiments indicate a 2:1 Hfq6/RNA stoichiometry for the strong binding complexes of Hfq with either A18 or DsrADII and suggests that RNA-induced dimer formation of Hfq6 is a common feature of Hfq−RNA interactions.