No abstract

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in posttranscriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.Hfq protein from Escherichia coli was first described in connection with Qβ-phage replication 1,2 . Hfq has recently emerged as a central player in post-transcriptional gene regulation as mediated by bacterial ncRNAs [3][4][5][6] . Escherichia coli Hfq mutants show disrupted signaling in stress response pathways 7,8 , arising from the need for Hfq to mediate base-pairing between regulatory ncRNAs and their mRNA targets. Examples of these partnerships include DsrA-rpoS 7,9,10 , OxyS-fhlA 11,12 , OxyS-rpoS 13 , RprA-rpoS 14 , RyhBsodB [15][16][17] .Complexes between ncRNAs and their mRNA targets function in several ways. Most commonly, complexed structures lead to translational activation or repression by remodeling mRNA regulatory regions containing the ribosome-binding site (RBS) and/or start codon. Alternatively, the interaction can enhance decay of the target mRNA16 or simply block translation11. Clearly, Hfq facilitates base-pairing between ncRNAs and their targets, but how it does so is poorly understood. How the chaperone function relates to other Hfq activities such as the control of poly(A) tail elongation19 , 20 and regulation of mRNA stability21 , 22 is also unknown. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2011 April 5. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHfq shares sequence similarity to the eukaryotic Lsm proteins [23][24][25][26][27] We addressed these questions through a mutational analysis of Hfq, probing in vitro binding to several model RNAs that represent species with which Hfq must interact. Hfq mutants were assayed in vivo using a reporter assay and RNA lifetime experiments. Together, the results support a model wherein at least two independent RNA-binding sites exist on the Hfq hexamer, and juxtaposition of bound RNAs facilitates base-pairing. RESULTS Hfq mutagenesisTo identify amino acids essential for RNA binding, we constructed a series of E. coli Hfq misse...

No abstract

The reaction of nitric oxide with the carboxylate-bridged diiron(II) complex [Fe(2)(Et-HPTB)(O(2)CPh)](BF(4))(2) (1a) afforded the dinitrosyl adduct, [Fe(2)(NO)(2)(Et-HPTB)(O(2)CPh)](BF(4))(2) (1b), where Et-HPTB = N,N,N',N'-tetrakis(N-ethyl-2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane, in 69% yield. Compound 1b further reacts with dioxygen to form the bis(nitrato) complex, [Fe(2)(Et-HPTB)(NO(3))(2)(OH)](BF(4))(2) (1c). The structure of 1b was determined by X-ray crystallography (triclinic, P&onemacr;, a = 13.5765(8) Å, b = 15.4088(10) Å, c = 16.2145(10) Å, alpha = 73.656(1) degrees, beta = 73.546(1) degrees, gamma = 73.499(1) degrees, V = 3043.8(7) Å(3), T = -80 degrees C, Z = 2, and R = 0.085 and R(w) = 0.095 for 5644 independent reflections with I > 3sigma(I)). The two nitrosyl units are equivalent with an average Fe-N-O angle of 167.4 +/- 0.8 degrees. Spectroscopic characterization of solid 1b revealed an NO stretch at 1785 cm(-)(1) in the infrared and Mössbauer parameters of delta = 0.67 mm s(-)(1) and DeltaE(Q) = 1.44 mm s(-)(1) at 4.2 K. These data are comparable to those for other {FeNO}(7) systems. An S = (3)/(2) spin state was assigned from magnetic susceptibility studies to the two individual {FeNO} centers, each of which has a nitrosyl ligand antiferromagnetically coupled to iron. A least-squares fit of the chi vs temperature plots to a theoretical model yielded an exchange coupling constant J of -23 cm(-)(1), where H = -2JS(1).S(2), indicating that the two S = (3)/(2) centers are antiferromagnetically coupled to one another. An extended Hückel calculation on a model complex, [Fe(2)(NO)(2)(NH(3))(6)(O(2)CH)(OH)](2+), revealed that the magnitudes of Fe-N-O angles are dictated by pi-bonding interactions between the Fe d(xz)() and NO pi orbitals.

This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.