Cooperation of Escherichia coli Hfq hexamers in DsrA binding

Abstract

Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA and target mRNA. Hfq mediates DsrA-dependent translational activation of rpoS mRNA at low temperatures. rpoS encodes the stationary-phase s factor s S , which is the central regulator in general stress response. However, structural information on Hfq-DsrA interaction is not yet available. Although Hfq is reported to hydrolyze ATP, the ATP-binding site is still unknown. Here, we report a ternary crystal complex structure of Escherichia coli Hfq bound to a major Hfq recognition region on DsrA (AU 6 A) together with ADP, and a crystal complex structure of Hfq bound to ADP. AU 6 A binds to the proximal and distal sides of two Hfq hexamers. ADP binds to a purineselective site on the distal side and contacts conserved arginine or glutamine residues on the proximal side of another hexamer. This binding mode is different from previously postulated. The cooperation of two different Hfq hexamers upon nucleic acid binding in solution is verified by fluorescence polarization and solution nuclear magnetic resonance (NMR) experiments using fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with full-length Hfq and DsrA also supports cooperation of Hfq hexamers upon DsrA binding. The implications of Hfq hexamer cooperation have been discussed.

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3′ poly(U) tail

“…If this were the case, it would be possible that the proximal side of Hfq binds the terminator poly(U) tail, while the internal stem-loop/U-rich sequence could interact with the distal side or some other portion of the same Hfq molecule. In fact, it was demonstrated recently that a U-rich RNA oligonucleotide corresponding to the internal Hfq-binding site of DsrA interacts with not only the proximal site but also the distal site and the rim of the Hfq hexamer (Wang et al 2011). An alternative possibility would be that the poly(U) tail of one SgrS molecule is recognized by the proximal side of one Hfq hexamer, while the internal stem-loop/U-rich sequence binds the distal side of another Hfq hexamer as proposed recently for DsrA-Hfq interaction (Wang et al 2011).…”

“…In fact, it was demonstrated recently that a U-rich RNA oligonucleotide corresponding to the internal Hfq-binding site of DsrA interacts with not only the proximal site but also the distal site and the rim of the Hfq hexamer (Wang et al 2011). An alternative possibility would be that the poly(U) tail of one SgrS molecule is recognized by the proximal side of one Hfq hexamer, while the internal stem-loop/U-rich sequence binds the distal side of another Hfq hexamer as proposed recently for DsrA-Hfq interaction (Wang et al 2011). Structural studies on the Hfq-functional sRNA complexes are certainly required to clarify why the hairpin structure is needed for the efficient Hfq binding and how exactly Hfq binds to the Hfq-binding modules to facilitate base-pairing between sRNAs and target mRNAs.…”

Hfq binding changes the structure ofEscherichia colismall noncoding RNAs OxyS and RprA, which are involved in the riboregulation ofrpoS

“…The Hfq core has two distinct faces; these proximal and distal faces have both been shown to be involved in RNA binding. In particular, U-rich RNA sequences, often found in sRNAs, have been shown to interact with the proximal face of Hfq, while A-rich sequences, or A-R-N repeats (where R is a purine nucleotide and N is any nucleotide), usually found in mRNAs but also present in some sRNAs, have been shown to interact with the distal face (Schumacher et al 2002;Mikulecky et al 2004;Updegrove et al 2008;Link et al 2009;Sauer and Weichenrieder 2011;Wang et al 2011). Recently, sRNA binding to the lateral surface of the Hfq core has also been identified (Updegrove and War-tell 2011;Sauer et al 2012).…”

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

“…We were unable to identify any reasonable candidates in our crystallization condition that were likely to reside in this position and believe that this is simply an accumulation of spurious electron density along a pseudocrystallographic axis. It is worth noting, however, that in the higher resolution crystal structure of Ec Hfq bound to proximal face-bound AU 6 A (Wang et al 2011), a water coordinated metal ion was observed in a similar location and made contacts to the RNA phosphate backbone. Although this might still be the case for the Lm Hfq-U 6 complex, placement of a similarly hydrated metal ion is too speculative at the current resolution.…”

“…2). Further, in common among Hfq proteins is the hydrogen bond made between a conserved glutamine (Lm Hfq residue Q9; Ec [Wang et al 2011[Wang et al , 2013, St [Sauer and Weichenrieder 2011], and Sa [Schumacher et al 2002] Hfq residue Q8) and the exocyclic O2 atom of uracil. In all four proteins, the Oε of this glutamine typically hydrogen bonds to the side chain ε amino group of a conserved lysine residue (K58 in Lm Hfq, K57 in Sa Hfq, and K56 in Ec and St Hfq).…”