title: The stoichiometry of the Escherichia coli Hfq protein bound to RNA.
abstract: The Escherichia coli RNA binding protein Hfq is involved in many aspects of post-transcriptional gene expression. Tight binding of Hfq to polyadenylate sequences at the 3' end of mRNAs influences exonucleolytic degradation, while Hfq binding to small noncoding RNAs (sRNA) and their targeted mRNAs facilitate their hybridization which in turn effects translation. Hfq binding to an A-rich tract in the 5' leader region of the rpoS mRNA and to the sRNA DsrA have been shown to be important for DsrA enhanced translation initiation of this mRNA. The complexes of Hfq-A(18) 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-A(18) and Hfq-DsrA. In this work, mass spectrometry and analytical ultracentrifugation provide direct evidence that the strong binding mode of the Hfq hexamer (Hfq(6)) for A(18) and domain II of DsrA (DsrA(DII)) 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 were also consistent with 1:1 stoichiometry. Mass spectrometry of cross-linked samples of Hfq(6), A(18), and DsrA(DII) 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.
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
authors.forename: Taylor B
authors.initials: TB
authors.lastname: Updegrove
authors.forename: John J
authors.initials: JJ
authors.lastname: Correia
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Chen
authors.forename: Charles
authors.initials: C
authors.lastname: Terry
authors.forename: Roger M
authors.initials: RM
authors.lastname: Wartell
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D035001:Host Factor 1 Protein / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation
chemical_list: C405431:DsrA RNA, E coli; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated