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Structure of Escherichia Coli Hfq Bound to Polyriboadenylate RNA

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Structure of Escherichia Coli Hfq Bound to Polyriboadenylate RNA Structure of Escherichia coli Hfq bound to polyriboadenylate RNA Todd M. Linka, Poul Valentin-Hansenb, and Richard G. Brennana,1 aDepartment of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030-4009; and bDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark Edited by Carol A. Gross, University of California, San Francisco, CA, and approved September 24, 2009 (received for review August 3, 2009) Hfq is a small, highly abundant hexameric protein that is found in proteins that serve as iron reservoirs, including sodB (superoxide many bacteria and plays a critical role in mRNA expression and RNA dismutase), ftn (ferritin), and bfn (bacterioferritin). Hfq also stability. As an ‘‘RNA chaperone,’’ Hfq binds AU-rich sequences and functions in cell envelope integrity by binding the sRNAs MicA facilitates the trans annealing of small RNAs (sRNAs) to their target and RybB to down-regulate the expression of the outer mem- mRNAs, typically resulting in the down-regulation of gene expres- brane proteins (ompA, ompC, and ompW, respectively) during sion. Hfq also plays a key role in bacterial RNA decay by binding growth and environmental stress condition (23–26). tightly to polyadenylate [poly(A)] tracts. The structural mechanism Beyond its role as an RNA chaperone, Escherichia coli (Ec) by which Hfq recognizes and binds poly(A) is unknown. Here, we Hfq also functions in RNA stability by binding with nanomolar report the crystal structure of Escherichia coli Hfq bound to the to subnanomolar affinities to polyadenylate [poly(A)] tails that poly(A) RNA, A15. The structure reveals a unique RNA binding have been added to the 3Ј ends of RNAs by poly(A) polymerase mechanism. Unlike uridine-containing sequences, which bind to (PAP I) (27–29). Unlike cytosolic eukaryotic mRNA, the addi- the ‘‘proximal’’ face, the poly(A) tract binds to the ‘‘distal’’ face of tion of adenosine nucleotides to bacterial mRNAs enhances their Hfq using 6 tripartite binding motifs. Each motif consists of an degradation (30). The extent of poly(A) tailing of Ec mRNAs by adenosine specificity site (A site), which is effected by peptide PAP I has been estimated recently to be greater than 90% of all backbone hydrogen bonds, a purine nucleotide selectivity site (R ORFs of bacteria that are growing exponentially (31). Intrigu- site), and a sequence-nondiscriminating RNA entrance/exit site (E ingly, Hfq switches PAP I from a distributive to a processive site). The resulting implication that Hfq can bind poly(A-R-N) catalytic mode, allowing for the more extensive adenylation of BIOCHEMISTRY triplets, where R is a purine nucleotide and N is any nucleotide, was mRNAs (27). How Hfq causes this switch is unknown. Moreover, confirmed by binding studies. Indeed, Hfq bound to the oligori- Hfq protects poly(A) tails from degradation by the exoribo- bonucleotides (AGG)8, (AGC)8, and the shorter (A-R-N)4 sequence, nucleases RNase II and polynucleotide phosphorylase (PN- AACAACAAGAAG, with nanomolar affinities. The abundance of Pase), and the endoribonuclease RNase E (28). The latter two (A-R-N)4 and (A-R-N)5 triplet repeats in the E. coli genome suggests enzymes are members of the degradosome, a multicomponent additional RNA targets for Hfq. Further, the structure provides assembly that is responsible for RNA decay in bacteria (32). Hfq insight into Hfq-mediated sRNA-mRNA annealing and the role of has been shown to interact with polynucleotide phosphorylase Hfq in RNA decay. (PNPase), which adds AG-rich polynucleotide tails to the 3Ј ends of mRNAs that are terminated in a Rho-independent fashion, gene regulation ͉ poly(A) ͉ posttranscription regulation ͉ RNA binding ͉ and RNase E, the key endoribonuclease involved in RNA Sm/Lsm family destruction (27–29)—both presumably via protein-RNA-protein interactions (33). fq is a pleiotropic posttranscriptional regulator that is a A full understanding of the RNA binding mechanisms of Hfq Hmember of the Sm/Lsm superfamily and found in many and its role in the protection of poly(A) tails from degradation Gram-negative and Gram-positive bacteria (1–3). Hfq binds to from endo- and exoribonucleases has been hindered by the lack AU-rich stretches near stem loop structures of small noncoding of relevant high-resolution structures. Indeed, the structure of RNAs (sRNA) and their target mRNAs to facilitate the annealing Staphylococcus aureus (Sa) Hfq bound to the uridine-rich oligo- of their trans-encoded complementary sequences and hence to ribonucleotide (AU5G) is the only example of an Hfq-RNA effect message-specific translational regulation (4–8). Thus, Hfq complex (34). The uridines and adenosine of this stretch of has been called an ‘‘RNA chaperone.’’ In conjunction with these sRNA bind to the ‘‘proximal’’ face of Hfq circling the central sRNAs, Hfq appears to down-regulate the expression of the ma- pore and making base-specific contacts with multiple side chains. jority of targeted messages; however, some mRNAs, including rpoS, The uridine and adenosine binding sites are essentially identical. which encodes the stress response/stationary phase sigma factor ␴S, By contrast, binding and mutagenesis studies revealed that require Hfq for their efficient translation (4, 9, 10). poly(A) tails do not bind to the proximal face but rather to the opposite side of Hfq, the so-named ‘‘distal’’ face (35, 36). The In addition to its role in the ␴S stress response, Hfq regulates structural mode of poly(A) binding and recognition is com- the expression of multiple virulence factors and genes—which pletely unknown but critical for a full understanding of Hfq are located in pathogenicity islands in several pathogenic bacteria—and itself is considered a virulence factor (11–14). Hfq is also involved in the regulation of a quorum sensing in Author contributions: T.M.L. and R.G.B. designed research; T.M.L. and P.V.-H. performed Vibrio cholerae and Vibrio harveyi by eliciting the sRNA- research; T.M.L. and P.V.-H. contributed new reagents/analytic tools; T.M.L., P.V.-H., and mediated destabilization of the mRNAs that encode the R.G.B. analyzed data; and T.M.L., P.V.-H., and R.G.B. wrote the paper. quorum-sensing master regulators hapR and luxR, respectively The authors declare no conflict of interest. (15). Further, Hfq plays a key role in the cellular response to This article is a PNAS Direct Submission. phosphosugar toxicity and low cellular iron concentrations (16– Freely available online through the PNAS open access option. 22). To combat phosphosugar toxicity, ptsG, which encodes a Data deposition: Coordinates and structure factors have been deposited in the Research sugar transporter, is bound by the sRNA SgrS and degraded by Collaboratory for Structural Bioinformatics database, www.rcsb.org (RCSB ID code 3GIB). the principal cellular endoribonuclease RNase E in an Hfq- 1To whom correspondence should be addressed. E-mail: [email protected]. mediated process. In times of iron scarcity, Hfq and its sRNA This article contains supporting information online at www.pnas.org/cgi/content/full/ partner, RhyB, direct the destruction of mRNAs that encode 0908744106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908744106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 Fig. 1. The structure of Ec Hfq bound to poly(A) RNA. (A) Electron density maps for the RNA tripartite module. Composite Fo–Fc omit map densities are shown in blue (0.8␴) and 2FoϪFc densities are shown in white (1␴). A stick model of the distal-side binding A15 RNA is shown with carbons colored slate. Hfq is shown as a gray surface. (B) A ribbon diagram of the biologically relevant Hfq hexamer and A18 oligonucleotide superimposed onto the apo Hfq hexamer (green). The Hfq trimers and A9 oligonucleotides of the 2-fold related asymmetric units are gray and black, respectively. One of the 6 tripartite binding repeats is labeled A (adenosine site), R (purine nucleotide site), and E (entrance/exit site). (C) Side view. A stick model of the bound RNA on the distal side is shown with carbons colored white, with one tripartite binding unit colored bluish gray. A CPK model of AU5G RNA with green carbons is docked in the proximal pore, as seen in the Sa-Hfq-AU5G structure. Hfq is shown as a gray surface. Figures were produced with PyMol (64). function in RNA decay and posttranscription regulation. Here, The Tripartite (A-R-E) RNA Binding Motif. Hfq binds the poly(A) tail we report the structure of Ec Hfq bound to a poly(A) tail. The by sectioning the sequence into 6 trinucleotide repeats with the structure reveals a unique RNA binding mechanism that is well 5Ј-adenosine nucleotide binding to the adenosine specificity site suited for not only poly(A)-tract binding but also triplet repeats (A site), the following nucleotide to the purine nucleotide of the type (adenine-purine-any base). The mechanistic impli- selectivity site (R site; so named because of its likely ability to cations of this promiscuous binding site in the biological func- bind guanosine as well as adenosine), and the third nucleotide to tions of Hfq are discussed. a nondiscriminatory RNA entrance/exit site (E site; Fig. 1). Thus, the distal face of hexameric Hfq has the capacity to bind Results and Discussion 18 nucleotides. Global Structure of the Hfq-Poly(A) Complex. A C-terminal trunca- The specificity of Hfq for adenosine nucleotides is effected tion mutant of Ec Hfq, lacking residues 70–102, was crystallized primarily by the A site, which is a surface-exposed groove with the oligoribonucleotide A15. This truncation, although composed of residues from ␤-strands 2 and 4 (Fig.
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