The stoichiometry of the Escherichia coli Hfq protein bound to RNA

Abstract

The Escherichia coli RNA binding protein Hfq is involved in many aspects of post-transcriptional gene expression. Tight binding of Hfq to polyadenylate sequences at the 39 end of mRNAs influences exonucleolytic degradation, while Hfq binding to small noncoding RNAs (sRNA) and their targeted mRNAs facilitate their hybridization which in turn effects translation. Hfq binding to an A-rich tract in the 59 leader region of the rpoS mRNA and to the sRNA DsrA have been shown to be important for DsrA enhanced translation initiation of this mRNA. The complexes of Hfq-A 18 and Hfq-DsrA provide models for understanding how Hfq interacts with these two RNA sequence/structure motifs. Different methods have reported different values for the stoichiometry of Hfq-A 18 and Hfq-DsrA. In this work, mass spectrometry and analytical ultracentrifugation provide direct evidence that the strong binding mode of the Hfq hexamer (Hfq 6 ) for A 18 and domain II of DsrA (DsrA DII ) involve 1:1 complexes. This stoichiometry was also supported by fluorescence anisotropy and a competition gel mobility shift experiment using wild-type and truncated Hfq. More limited studies of Hfq binding to DsrA as well as to the sRNAs RprA, OxyS, and an 18-nt segment of OxyS were also consistent with 1:1 stoichiometry. Mass spectrometry of crosslinked samples of Hfq 6 , A 18 , and DsrA DII exhibit intensity corresponding to a ternary 1:1:1 complex; however, the small intensity of this peak and fluorescence anisotropy experiments did not provide evidence that this ternary complex is stable in solution.

Cooperation of Escherichia coli Hfq hexamers in DsrA binding

“…A differnce in binding stoichiometries observed in various methods and experiment conditions has been reported. A 2:1 Hfq 6 :DsrA binding ratio has been observed at submicromolar Hfq 6 concentrations (Lease and Woodson 2004;Mikulecky et al 2004;Sun and Wartell 2006), while 1:1 stoichiometry has also been reported (Brescia et al 2003;Hwang et al 2011;Updegrove et al 2011). Our knowledge is still very limited due to the lack of atomic resolution structural information.…”

“…1A) is believed to bind to the proximal side on Hfq (Brescia et al 2003;Updegrove et al 2011), our crystal structure suggests that AU 6 A (28;35 nt on DsrA), which is a part of DsrA DII , inserts its 59-end A28 into the R site on the distal face of another Hfq hexamer (Fig. 3A).…”

“…The affinity of CU 6 C and GU 6 G with Hfq is moderately higher than AU 6 A, indicating a small effect of flanking bases on the affinity of the U-rich segment with Hfq. Actually, Hfq has been reported to associate with the 39 end as well as internal U-rich stretches with variations in the U-rich sequence length and interleaving or flanking nucleotide bases (Lease and Woodson 2004;Sun and Wartell 2006;Salim and Feig 2010;Hopkins et al 2011;Sauer and Weichenrieder 2011;Updegrove et al 2011;Vogel and Luisi 2011). The variation of the U-rich RNA recognition in the Hfq complex structures suggests that the differences in nucleoside-binding sites, the number of occupied binding pockets, and the phosphoribose conformations may be important in the recognition of different U-rich substrates and modulation of binding affinity.…”

“…AU 6 A is a major U-rich Hfq-binding site on the linker between DsrA SLI and SLII that was previously reported to associate with the Hfq proximal side (Lease and Woodson 2004;Mikulecky et al 2004;Sun and Wartell 2006;Updegrove et al 2011). AU 6 A is different from the canonical U-rich sequence AU 5 G recognized by Sm proteins.…”

Duplex formation between the sRNA DsrA andrpoSmRNA is not sufficient for efficient RpoS synthesis at low temperature

“…19 In vivo DsrA·rpoS duplex formation at low temperature further requires the CsdA helicase, 20 and creates RNase III cleavage sites within the duplex that prevents reuse of DsrA. 21 DsrA was shown to bind with a 1:1 stoichiometry on the proximal face of Hfq, 8,22 whereas rpoS mRNA is recruited to At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor Rpos, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs).…”