Structural Studies Of E.coli RNA Chaperon Hfq And Its Interactions With RNA

Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA andtarget mRNA. Hfq mediates DsrA dependent translational activation of rpoS mRNA at lowtemperatures. rpoS encodes the stationary phase sigma factorσSwhich is the central regulatorin general stress response. E.coli Hfq is a 102 amino acid residue (11.2kDa) bacterial SmRNA chaperon protein. The N-terminal 1~65 amino acid residues of Hfq protein (Hfq65)comprise a conserved Sm domain and the C-terminal part 66~102aa (HfqCTD) isnon-structured but functional essential. The Sm domain forms homo-hexameric ring structure.The molecular weights for Hfq65 and Hfq full length hexamers are 43kDa and 67kDarespectively. Distinct RNA binding sites with specificity for A-rich and U-rich ssRNArespectively has been identified on either side of the ring. However, structural information onHfq-DsrA interaction is not yet available. Though Hfq is reported to hydrolyze ATP, neitherthe ATP binding site nor the biological significance of ATP hydrolysis is known. Moreover,the structure and function of the flexible tail of HfqCTD remains an enigma. We haveassigned backbone resonances for Hfq65 and HfqCTD and acquired I, L, V methyl selectivelylabeled Hfq65 and Hfq full length (HfqFL) HMQC spectra. We found that Hfq65 and HfqFLbehaves very differently on both1H-15N HSQC and1H-13C methyl HMQC. Mutual NMRtitrations of HfqCTD with Hfq65 indicate non-prominent interaction between these twoconstructs. We have used Gd(DPA)3as paramagnetic probe and acquired preliminaryparamagnetic relaxation enhancement (PRE) results suggesting transient interactions betweenHfq65 and HfqCTD in HfqFL protein. We have also acquired ternary crystal complexstructure of E.coli Hfq bound to a major Hfq recognition region on DsrA, AU6A, togetherwith ADP and crystal complex structure of Hfq bound to ADP. AU6A binds to proximal anddistal sides of two Hfq hexamers. ADP bind to purine selective site on distal side and contactconserved arginine or glutamine residues on proximal side of another hexamer. This bindingmode is different from previously postulated. In ATPase activity assays, we confirmed thatthe residues that make contact with ADP is involved in the ATPase activity. We found thatATPase activity decreases the binding affinity of Hfq to U-rich sequence. In cases when Hfqconcentration is much lower than RNA concentration, the presence of ATP can increase thechaperoning ability of Hfq. The cooperation of two different Hfq hexamers upon nucleic acidbinding in solution is verified by fluorescence polarization and solution NMR experimentsusing fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with fulllength Hfq and DsrA also support cooperation of Hfq hexamers upon DsrA binding. Weproposed a plausible model for Hfq mechanism in which cooperation between multiple Hfqhexamers is required.