Browsing by Author "Brennan, Richard G"

As bacterial resistance to multiple antibiotics continues to become a growing problem across the globe, the imperativeness for understanding mechanisms of antibiotic and multidrug resistance is increasingly apparent. Currently, the gram-negative bacteria Neisseria gonorrhoeae and Escherichia coli are considered urgent public health threats due to the rise in multidrug resistant strains. Mechanisms by which these bacteria become resistant to antibiotics include the overexpression of multidrug efflux systems. Prior to the introduction of antibiotics, the primary purpose of these multidrug efflux systems was to protect the bacteria from cytotoxins in the environment including innate host defense molecules or toxic molecules produced by the bacteria. Overtime, the bacteria have adapted these efflux systems to protect against clinically relevant antibiotics used to clear these bacterial infections. These multidrug efflux systems are energetically expensive to synthesize; thus, they are often tightly regulated at the transcription level by transcription repressors or activators. Many multidrug efflux systems are regulated by transcriptional regulators with proximal genes that specifically regulate a single multidrug efflux system. However, the expression of a few multidrug efflux systems is controlled by unique repressors that act as global regulators, which have a larger role in regulating complex virulence and stress response systems. Specifically, examples of multidrug efflux regulators in N. gonorrhoeae and E. coli that act as global regulators within their respective genomes include N. gonorrhoeae MtrR and E. coli MprA. Understanding the global regulatory activities of these two transcription regulators will broaden our understanding of the regulatory mechanisms that enable bacterial survival during host infection, the mechanisms that contribute to antibiotic resistance, as well as the fundamentals of bacterial transcription regulation. To provide novel insight into the global regulatory activities and function of N. gonorrhoeae MtrR, this dissertation expounds a series of original structural, biochemical, and in vivo studies identifying cytotoxin and DNA recognition mechanisms of MtrR. Previous work showed that MtrR represses directly the mtrCDE efflux transporter genes by binding an operator between the mtrR and mtrC genes; additionally, MtrR represses directly the rpoH oxidative stress response sigma factor. MtrR-mediated repression of the mtrCDE genes had been shown to be relieved upon exposure of gonococci to toxic hydrophobic agents and detergents (i. e. MtrR is “induced” by these toxic molecules). However, physiologically relevant innate host molecules recognized by MtrR had not been identified. In this work, we identify bile salts present at extra-urogenital gonococcal infection sites that MtrR directly binds, to result in derepression of the mtrCDE genes in vitro and in vivo. Furthermore, we use x-ray crystallography to solve structures of MtrR in its induced form and bound to the mtrCDE and rpoH operators. With these structures, we determined the structural mechanism of induction of MtrR. In addition, the MtrR-operator structures reveal a degenerate consensus sequence to which MtrR binds within the mtrCDE and rpoH operators. Mechanisms for cytotoxin and DNA recognition were confirmed by structure-guided site-directed mutagenesis studies and a combination of biochemical binding assays utilizing isothermal titration calorimetry (ITC) or fluorescence polarization (FP). Importantly, this structural and biochemical work also reveals the mechanisms by which common mutations in multidrug resistant strains of N. gonorrhoeae confer resistance. To elucidate the function of E. coli MprA and realize its potential as a drug target, this dissertation also includes research describing the ligand-binding mechanisms of MprA. MprA (formerly EmrR) represses directly the EmrAB efflux pump in E. coli. Previously published work identified MprA as the molecular target of a small molecule inhibitor (DU011) of the biosynthesis of an important virulence factor in E. coli, the polysaccharide capsule. This lead molecule has the potential for optimization for drug development and reveals a novel function of MprA as a regulator of polysaccharide capsule synthesis. We characterized the interaction between MprA and DU011 and compared this to the binding between MprA and other previously identified ligands including salicylate and 2,4-dinitrophenol (DNP) utilizing ITC assays. Through these studies, we revealed a novel binding mode for MprA and laid the groundwork for future structural studies and drug optimization. Collectively, this work provides important insight into the breadth of regulatory functions of N. gonorrhoeae MtrR and E. coli MprA, two key global regulators from highly prevalent multidrug resistant pathogens. Specifically, the original research presented here provides a biochemical evaluation of the bacterial stress response mechanisms controlled by MtrR and MprA and their contribution to antibiotic resistance. Indeed, the biochemical and structural characterization of these two regulators will inform future work to combat multidrug resistance.

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

Abstract

Hfq is a pleiotropic posttranscriptional regulator and RNA chaperone that facilitates annealing of trans-encoded sRNA/mRNA pairs. It regulates many different cellular pathways including environmental stress responses, quorum sensing, virulence and maintenance of membrane integrity. Hfq is a member of the Sm/LSm family and forms a homohexamer that has two faces, termed proximal and distal. Hfq preferentially binds A/U rich regions that are near stem loop structures. Crystal structures have shown that poly-A sequences tend to bind the distal face while poly-U sequences bind the proximal face. Currently crystal structures reveal the binding mechanisms for short RNA sequences however; physiologically relevant RNA sequences are typically longer and more structured. To study how these more complex RNA sequences interact with Hfq, a tryptophan fluorescence quenching (TFQ) assay has been developed. Here it is presented that TFQ can correctly identify the binding face for two control sequences, A15 and U6, using the E. coli, S. aureus and L. monocytogenes Hfq homologues. Using fluorescence anisotropy and crystallography it is observed that Trp mutants necessary for TFQ may affect binding to some degree but do not affect the overall structure or RNA binding function of Hfq. TFQ is then used to examine the distal face binding motifs for both Gram-negative (E. coli) and Gram-positive (S. aureus/L. monocytogenes) Hfq, (A-R-N)n and (R-L)n respectively. Using sequences that either fulfilled just (A-R-N)n or both (A-R-N)n and (A-A-N)n motifs it is shown that the distal face motif for Gram-negative Hfq is the more specific (A-A-N)n motif. Using sequences that either fulfilled just (R-L)n or both (R-L)n and (A-L)n motifs it is shown that the Gram-positive distal face motif can be redefined to the (A-L)n motif. Finally TFQ is used to explore autoregulation of E. coli hfq. Two identified binding sites located in the 5'UTR of hfq mRNA, site A and site B, were used for TFQ, along with a longer RNA sequence that contains both sites and their native linker, 5' UTR. TFQ illustrates that the individual sites and the 5' UTR are capable of binding both faces. Each site appears to prefer binding to one face over the other, suggesting a model for hfq 5' UTR mRNA binding to Hfq where either one or two hfq mRNA bind a single Hfq hexamer. In conclusion, TFQ is a straightforward method for analyzing how RNA sequences interact with Hfq that can be utilized to study how longer, physiologically relevant RNA sequences bind Hfq.

Multidrug tolerance (MDT) is the ability of pathogenic bacteria to survive killing from exposure to multiple antibiotics, and is a major obstacle in the treatment of infectious disease. A small population of bacteria (0.0001%) termed persisters is the culprit that causes MDT and allows these cells to persist. In Escherichia coli, the HipBA toxin–antitoxin pair plays a role in multidrug tolerance. HipA, a 50 kDa serine protein kinase, is the more stable toxin and abrogates cell growth in the absence of the more labile antitoxin HipB. HipB is a transcription repressor that binds to the four conserved (TATCCN₈GGATA) operator sites of the hipBA promoter to autoregulate expression of the hipBA operon. Delineation of the molecular mechanism of HipB–hipBAoperator binding is critical to understand fully the regulation of persistence by HipB. Thus, we determined the equilibrium dissociation constants (K_d) of HipB for each of the four hipBA operators and the paired operator sites O₁O₂ and O₃O₄. We found that the affinity of HipB for binding the O₁ and O₃ operators is seven to eight times higher than for the O₂ and O₄ operators. In addition, the affinity of HipB for the O₁O₂ and O₃O₄ operators is at least four times higher than the O₁ and O₃ operators. The HipB–operator complex structures reveal that HipB makes the same key contacts to the conserved TATCC motifs and bends each operator DNA by the same extent between 50° to 70° implying thus the affinity differences are attributed to indirect readout of the 8 base pair spacer (N₈). Mutational studies on residues involved in HipB–DNA interaction revealed the contribution of a series of selected residues to binding affinity with residues K38 and Q39 contributing greatly to affinity whereas other base contacting residues S29 and A40 contribute less to affinity. Surprisingly residue S43, which is involved in a hydrogen bond to the DNA phosphate backbone contributes more than expected because S43 forms a hydrogen bond network with nearby water molecules.

HipA was the first described bona fide persistence factor. The hip locus was discovered through a mutagenesis screen whereby hipA7 was isolated. Described herein, biochemical and structure–function studies on HipA7, the gene product of the high persistent mutant allele having two point mutations G22S and D291A, revealed that the D291A mutation weakens the binding affinity for HipB by 3 to 4 fold. The HipA7 structure revealed the conformational heterogeneity of the P–loop motif (the ATP binding motif), which suggests a dynamic role of the loop in regulation of the kinase activity of HipA. To identify in vivo HipA substrates, we developed a mass spectrometry (MS)–based kinase assay, which led to identification of a novel phosphorylation site (residue S348) on HipA and a proposed consensus phosphorylation motif +ϕS, where +, φ and S designate a positive, hydrophobic and serine amino acid residue, respectively. Phosphorylation of peptides with this consensus motif, including the S150 (EENDFRISVAGAQEK), S348 (TGIHISDLK) and GltX (GKKLSKRH), was confirmed subsequently by the MS–based kinase assay. Further analysis of the HipA7 structure suggested that HipA might undergo pyrophosphorylation on residue S150, and the MS–based kinase assay confirmed pyrophosphorylation of HipA.

Thus, our data support that HipA is a persistence factor via its kinase activity and precise hipBA gene regulation through HipB binding tightly to O₁ and O₃ is critical for the survival of bacteria in the presence of antibiotics. In addition, we propose a consensus motif for HipA substrates.

NatA binds inositol hexakisphosphate and other ligands, and exhibits conformational flexibility dependent on the ligand bound.

Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase pathogenicity island (FPI) that is regulated by the macrophage growth locus protein A (MglA), the stringent starvation protein A (SspA) and the pathogenicity island gene regulator (PigR). MglA•SspA•PigR interacts with the RNA polymerase (RNAP) to activate FPI transcription. The activity of MglA•SspA•PigR•RNAP is mediated by ppGpp, the alarmone of the stringent response. However, the molecular mechanisms involved in FPI transcriptional regulation are not well understood.

While most bacterial SspA proteins interact with the RNAP as a homodimer, F. tularensis SspA forms a heterodimer with MglA, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family, and unexpectedly formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that, while it can self-associate in solution to form a dimer, MglA preferentially forms a heterodimer with SspA. As research with MglA highlighted that MglA•SspA is likely the only functional SspA protein in Francisella, X-ray crystallography was used to determine the structure of MglA•SspA to 2.65 Å. Analysis of the MglA•SspA structure revealed a vast hydrogen bond network at the interface that is significantly larger than the network at the MglA homodimerization interface. This difference could explain why MglA and SspA form a stable heterodimer and MglA (in the absence of SspA) forms a transient homodimer.

To gain insight into the molecular mechanism by which ppGpp mediates FPI transcription, differential radial capillary action of ligand assay (DRaCALA) was performed to determine which Francisella protein binds ppGpp. The DRaCALA experiments revealed that the MglA•SspA complex binds ppGpp specifically and with high affinity, while PigR, MglA and E. coli SspA do not. To characterize this interaction the structure of MglA•SspA was determined in complex with ppGpp (MglA•SspA•ppGpp) to 2.8 Å resolution, and revealed a single ppGpp molecule, which is also coordinated by Mg2+, bound per heterodimer. Analysis of structure-guided MglA•SspA ppGpp-binding mutants by DRaCALA confirmed the crystallographic binding site of ppGpp. Intriguingly, the binding of ppGpp does not produce a marked conformational change in MglA•SspA, but as the ppGpp-binding site overlaps with the previously ascribed PigR interaction surface it seems likely that ppGpp mediates the interaction between MglA•SspA and PigR.

The interaction between PigR and MglA•SspA is largely uncharacterized. Using fluorescence anisotropy we sought to determine which portion of PigR is responsible for interactions with MglA•SspA. Using fluoresceinated PigR peptides we determined that the final 22 residues of the PigR C-terminus interact with MglA•SspA. These results were corroborated using bridge-hybrid experiments by our collaborators. Further, as FA performed with and without ppGpp shows no difference in affinity between MglA•SspA and PigR, ppGpp must mediate this interaction through an as of yet undetermined mechanism, which is supported by very different millipolarization values indicating perhaps alternative binding modes of PigR.

All together, our results contribute significantly to our understanding of FPI transcriptional regulation.

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Acyl-carrier-protein (ACP) is the principal transporter of fatty acids, coordinating acyl-transfer among a vast network of diverse enzymes and biochemical processes. ACP association with protein partners is thought to be exceedingly transient. This paradigm has posed challenges for understanding the molecular basis for acyl-delivery and dissociation. During biosynthesis of the lipid A component (endotoxin) of lipopolysaccharides, ACP shuttles acyl-intermediates thioester-linked to its 4'-phosphopantetheine arm among four acyltransferases: LpxA, LpxD, LpxL, and LpxM. LpxA and LpxD are essential cytoplasmic enzymes, which not only provide an excellent model system to study ACP-based interaction, but also offer an important therapeutic target for development of novel antibiotics. The current dissertation reports the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and breakage products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release. Moreover, the crystal structure of E. coli LpxA in complex with one form of ACP (holo-ACP) is presented. The structure reveals three molecules of holo-ACP localize to the C-terminal domain of the LpxA homotrimer, and shows the functional role of this domain is two-fold: ACP recognition and nucleotide binding of UDP-GlcNAc. A comparison with the LpxD:ACP complexes uncovers that ACP utilizes different surface residues for recognition even amongst closely related acyltransferases, yet still relies on "electrostatic steering" for docking to its enzyme partner. Insights gleaned from the presented structures have provided not only a better understanding of ACP interaction with acyltransferases, but also has identified the "drugable molecular landscape" for the development of novel antibiotics against infective bacteria.

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

The bacterial protein Hfq is an RNA chaperone and pleiotropic posttranscriptional regulator. Hfq binds to A and U-­‐‑rich regions of small regulatory RNA (sRNA) to their cognate mRNA to facilitate their annealing, affecting stability and translation. The protein is involved in the regulation of a wide array of cellular processes, including many related to environmental stress response and virulence. The importance of Hfq in Gram-­‐‑negative bacteria is well understood, while a less clear picture remains for Gram-­‐‑positive species. We have determined the structure of Hfq from the Gram-­‐‑positive pathogen Listeria monocytogenes (Lm) in its apo form and bound to U6 RNA. U6 RNA binds to the proximal face in a canonical manner but with additional contacts made to the N3 and O4 positions of uridine by residue Q6 of Hfq. Furthermore, fluorescence polarization and tryptophan fluorescence quenching (TFQ) reveal that U16 RNA binds to Hfq with higher affinity than U6, on the basis of the longer sequence’s ability to simultaneously bind in the proximal pore and the lateral rim of the protein. TFQ also shows that surprisingly Lm Hfq can accommodate (GU)3G and U6 RNA on both proximal and distal face binding sites, suggesting Lm Hfq has a less stringent distal face A-­‐‑site than previously reported for Hfq from other species.

To understand fully how sRNA bind to the proximal face and are positioned to anneal with mRNA, we have attempted to crystallize U16 RNA with Lm Hfq and fragments of sRNA containing a hairpin with a poly-­‐‑U tail with both Lm and Escherichia coli (Ec) Hfq. While this endeavor has been largely fruitless, we have determined the structure of Ec Hfq with dsDNA. Ec Hfq-­‐‑DNA binding has been observed in multiple studies but the molecular mechanism of recognition of this nucleic by Hfq is unknown. The DNA binds to the proximal face with conserved lateral rim residues N13, R16, and R17, and residue Q41 contacting the phosphate backbone. Fluorescence polarization and TFQ reveal both dsDNA and dsRNA bind to the proximal face, indicating the observed DNA binding mode may actually be a double stranded nucleic acid binding site. We have thusly proposed a model in which the proximal face of Hfq stabilizes both single and double stranded portions of sRNA, positioning it appropriately for formation of an Hfq-­‐‑sRNA-­‐‑mRNA ternary complex.

Toxin-­‐‑antitoxin (TA) modules are ubiquitous among bacterial species with bioinformatics studies identifying at least 10000 putative TA modules. These modules have diverse functions and are implicated in many processes, including gene regulation, stress response, and persister cell formation. Whereas many bacteria may have only a handful of TA modules, the genome of Mycobacterium tuberculosis (Mtb) contains 79 TA modules, 37 of which have been confirmed to be functional in vivo. A recent transcriptome analysis of Mtb persister cells revealed 10 up-­‐‑regulated TA modules. Four of these modules do not belong to a previously characterized TA family. We have determined the structure of a C-­‐‑terminally truncated version of the toxin Rv3189 (1-­‐‑164 of 206 amino acid residues) in complex with the anti-­‐‑toxin Rv3188. Rv3189 is structurally homologous to the ADP-­‐‑ribosyltransferase core domain, suggesting a never before observed mode of action for a TA module. The toxin has been shown to inhibit growth in an E. coli model with structure guided mutagenesis identifying residues that are critical for toxin function.