Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites

被引:61
作者
Sun, XG [1 ]
Wartell, RM [1 ]
机构
[1] Georgia Inst Technol, Sch Biol, Atlanta, GA 30322 USA
关键词
D O I
10.1021/bi0523613
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (DsrA(DII)) and A(18) 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 A(18) (<= 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 DsrA(DII) by 6-8-fold, while other mutations on the distal or proximal surfaces affected affinity to DsrA(DII) by <= 2-fold using the gel-mobility shift assay. The F39A/L12F double mutation partially regained the affinity for DsrA(DII) 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 Hfq(6)/RNA stoichiometry for the strong binding complexes of Hfq with either A18 or DsrA(DII) and suggests that RNA-induced dimer formation of Hfq(6) is a common feature of Hfq-RNA interactions.
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页码:4875 / 4887
页数:13
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