Defining the structural mechanisms of Hfq binding to cognate nucleic acids

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DESCRIPTION (provided by applicant): Hfq is a pleiotropic, posttranscriptional regulator found in many bacteria. Hfq plays a critical role in the cellular response to multiple stresses. Hf is also a bona fide virulence factor and contributes to multidrug resistance. As part of its function, Hfq binds A/U-rich sequences to facilitate the annealing of small RNAs (sRNAs) to target mRNAs, typically repressing their translation. Hence Hfq is an RNA-chaperone. Hfq also alters the stability of sRNAs and Escherichia coli (Ec). A full understanding of Hfq function has been hampered in great part by the dearth of high-resolution structures of germane Hfq-RNA complexes. Although the reported structures of Hfq bound to smaller oligoribonucleotides reveal that Hfq uses two faces to bind RNA, they leave multiple mechanistic and functional questions unanswered. Hfq is also found in the nucleoid and binds DNA. The Hfq- DNA binding mechanism is a mystery and structural studies are needed to unravel it. Perhaps the two key questions remaining are: how does Hfq bind to larger, mRNA targets, sRNAs and sRNA-mRNA complexes and is there a simple or even complex sequence or structure-based code that Hfq uses to bind DNA (or even RNA)? The answers to these questions are crucial to a complete understanding of Hfq function. Hence with these big questions in mind, this high risk-high reward R21 grant proposal has two Specific Aims that employ primarily crystallography. The first Specific Aim is to determine the structures of Hfq from Gram-negative and Gram-positive bacteria bound to physiologically relevant cognate sRNAs, mRNAs and their ternary complexes. As a component of this Aim, tryptophan fluorescence quenching experiments using a series of single tryptophan-containing Hfq proteins from E. coli and L. monocytogenes will provide an initial, guiding lower- resolution map of the RNA binding sites on each protein. The second Specific Aim is to characterize biochemically and structurally Ec Hfq-DNA complexes using intrinsically curved dsDNA sequences, other dsDNA sequences, and single stranded A-tract DNA and to identify chromosomally encoded Hfq-DNA binding sites using REPSA and ChAP-seq. The longer-term goal of this research is to delineate the structural and biochemical mechanisms that Hfq uses to function as a posttranscriptional regulator and possibly as a nucleoid-associated transcriptional regulator. Since the loss of Hfq attenuates bacterial virulence, this protein is a potential target for chemotherapeutic intervention, and the proposed structures of biologically relevant Hfq- RNA complexes will provide invaluable guidance in any future drug design efforts.