Binding properties of Hfq to RNA and genomic DNA and the functional implications

Hfq, also known as Host Factor I, is an 11.2 Kilo-Dalton heat stable protein that is a required host factor for bacteriophage Qbeta RNA replication in E. coli. Early studies have shown Hfq protein to be a global regulator of E. coli metabolism, which can be seen in the pleiotropic phenotypes of Hfq knockout mutants; E. coli Hfq mutants fail to respond to various stress insults, thus leaving the bacterium vulnerable. The broad impact of this protein appears to stem from its role in regulating the stability and/or translation of mRNA from a number of regulatory genes in an array of bacterial species.;The interaction of E. coli Hfq with RprA and two portions of the rpoS mRNA leader region was examined to explore Hfq's effect on promoting RprA-rpoS RNA binding in vitro . One rpoS RNA, rpoS-L, contained the entire 565-nucleotide untranslated leader region, while the other, rpoS-S, contained the 199-nucleotide sequence surrounding the start codon. An RNase H assay indicated both rpoS RNAs have similar secondary structures in the translation initiation region. Hfq formed two complexes with RprA in a gel mobility assay with binding parameters similar to values previously determined for DsrA. Unlike DsrA, Hfq binding to RprA was inhibited by poly(A) and influenced by Hfq mutations on both the distal and proximal surfaces. Hfq increased the level of RprA binding to both rpoS RNAs but showed a much larger enhancement when rpoS -L was examined. The lower affinity of RprA for rpoS-L versus rpoS-S in the absence of Hfq suggests that Hfq overcomes an inhibitory structure within rpoS-L in stimulating RprA binding. Similar results were obtained with DsrA. The results indicate that the full upstream leader sequence of rpoS mRNA influences Hfq-facilitated annealing of RprA and DsrA and is likely to be involved in its regulation.;The sRNAs DsrA and RprA enhance translation of rpoS mRNA by pairing to a site on this mRNA and disrupting an intramolecular stem-loop structure containing the ribosome binding site (RBS). The sRNA OxyS represses rpoS mRNA translation by an unknown mechanism. The binding of eleven mutant Hfqs to DsrA, RprA, OxyS, and two segments of the rpoS mRNA untranslated leader region was examined to explore RNA binding surfaces on Hfq. Mutant Hfqs were also tested for their ability to stimulate DsrA- rpoS RNA binding. Nine of the mutant Hfqs had single amino acid mutations located on the proximal, distal, or outer-circumference surface of the Hfq hexamer structure. Two mutant Hfqs had truncated C-terminal ends. Proximal surface mutations decreased Hfq binding to the three sRNAs and the rpoS RNA segment containing the RBS.;Hfq is involved in many aspects of posttranscriptional gene expression. Tight binding of Hfq to polyadenylate sequences at the 3' end of mRNAs influences exonucleolytic degradation, while Hfq binding to sRNAs and their targeted mRNAs facilitate their hybridization which in turn effects translation. Hfq binding to the sRNA DsrA and to an A-rich tract in the 5' leader region of the rpoS mRNA have been shown to be important for DsrA enhanced translation initiation of this mRNA. The complexes of Hfq-A18 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-A18 and Hfq-DsrA. In this work, mass spectrometry and analytical ultracentrifugation were utilized to provide direct evidence that the strong binding mode of the Hfq hexamer (Hfq6) for A18 and domain II of DsrA ((DsrADII), a 38-nt portion of DsrA that competes with full length DsrA for Hfq binding), 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 that binds Hfq were also consistent with 1:1 stoichiometry. Mass spectrometry of a sample containing Hfq6, A18, and DsrADII 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. (Abstract shortened by UMI.)...