title: Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.
abstract: In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)(15) and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65)) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Hermann
authors.initials: H
authors.lastname: Hämmerle
authors.forename: Mads
authors.initials: M
authors.lastname: Beich-Frandsen
authors.forename: Branislav
authors.initials: B
authors.lastname: Večerek
authors.forename: Lukas
authors.initials: L
authors.lastname: Rajkowitsch
authors.forename: Oliviero
authors.initials: O
authors.lastname: Carugo
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.forename: Udo
authors.initials: U
authors.lastname: Bläsi
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D035001:Host Factor 1 Protein / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D009695:Nucleic Acid Renaturation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D008024:Ligands; D018832:Molecular Chaperones; D021122:Protein Subunits; D012329:RNA, Bacterial; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases