Ross A. Edwards

FinO is an RNA chaperone that facilitates sense–antisense RNA interactions

The protein FinO represses F‐plasmid conjugative transfer by facilitating interactions between the mRNA of the major F‐plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem–loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense–antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense–antisense pairing. The energy for FinO‐catalyzed base‐pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA‐binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense–antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

Comparison of the Structures and Peptide Binding Specificities of the BRCT Domains of MDC1 and BRCA1

The tandem BRCT domains of BRCA1 and MDC1 facilitate protein signaling at DNA damage foci through specific interactions with serine-phosphorylated protein partners. The MDC1 BRCT binds pSer-Gln-Glu-Tyr-COO(-) at the C terminus of the histone variant gammaH2AX via direct recognition of the C-terminal carboxylate, while BRCA1 recognizes pSer-X-X-Phe motifs either at C-terminal or internal sites within target proteins. Using fluorescence polarization binding assays, we show that while both BRCTs prefer a free main chain carboxylate at the +3 position, this preference is much more pronounced in MDC1. Crystal structures of BRCA1 and MDC1 bound to tetrapeptide substrates reveal differences in the environment of conserved arginines (Arg1699 in BRCA1 and Arg1933 in MDC1) that determine the relative affinity for peptides with -COO(-) versus -CO-NH(2) termini. A mutation in MDC1 that induces a more BRCA1-like conformation relaxes the binding specificity, allowing the mutant to bind phosphopeptides lacking a -COO(-) terminus.

Structural basis of specific TraD–TraM recognition during F plasmid-mediated bacterial conjugation

F plasmid‐mediated bacterial conjugation requires interactions between a relaxosome component, TraM, and the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore. Here we present the crystal structure of the C‐terminal tail of TraD bound to the TraM tetramerization domain, the first structural evidence of relaxosome‐coupling protein interactions. The structure reveals the TraD C‐terminal peptide bound to each of four symmetry‐related grooves on the surface of the TraM tetramer. Extensive protein–protein interactions were observed between the two proteins. Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. Our results suggest that specific interactions between the C‐terminal tail of TraD and the TraM tetramerization domain might lead to more generalized interactions that stabilize the relaxosome‐coupling protein complex in preparation for conjugative DNA transfer.

ProQ is an RNA chaperone that controls ProP levels in Escherichia coli.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

Molecular insights into the function of RING finger (RNF)-containing proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent ubiquitylation.

Background: RNF8 and RNF168 are essential RING-E3 ubiquitin ligases that catalyze the formation of Lys-63 ubiquitin chains in the DNA damage response (DDR). Results: We solved the crystal structures and probed the activity of RNF8 and RNF168 in vitro. Conclusion: RNF168 likely acts downstream of RNF8 given its deficient activity. Significance: Our data provide structural and catalytic insight into the relative activities of RNF8 and RNF168. The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.

Silencing of natural transformation by an RNA chaperone and a multitarget small RNA

Significance Natural transformation is a major mechanism of horizontal gene transfer (HGT) by which bacteria take up exogenous DNA directly in their environment and integrate it in their genome. Acquiring new genetic information may confer an adaptive advantage but an uncontrolled uptake of foreign DNA may be harmful. We document a previously unsuspected means to control HGT by natural transformation in the human pathogen Legionella pneumophila. We found that the DNA uptake system required for natural transformation is subjected to silencing. A member of the widespread ProQ/FinO domain-containing protein family acts as an RNA chaperone and allows the targeting of the mRNAs of the genes coding the DNA uptake system by a newly identified trans-acting small RNA. A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila. We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

Structure of the Periplasmic Stress Response Protein CpxP.

CpxP is a novel bacterial periplasmic protein with no homologues of known function. In gram-negative enteric bacteria, CpxP is thought to interact with the two-component sensor kinase, CpxA, to inhibit induction of the Cpx envelope stress response in the absence of protein misfolding. CpxP has also been shown to facilitate DegP-mediated proteolysis of misfolded proteins. Six mutations that negate the ability of CpxP to function as a signaling protein are localized in or near two conserved LTXXQ motifs that define a class of proteins with similarity to CpxP, Pfam PF07813. To gain insight into how these mutations might affect CpxP signaling and/or proteolytic adaptor functions, the crystal structure of CpxP from Escherichia coli was determined to 2.85-Å resolution. The structure revealed an antiparallel dimer of intertwined α-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold, with the conserved LTXXQ motifs forming two diverging turns at one end. Biochemical studies demonstrated that CpxP maintains a dimeric state but may undergo a slight structural adjustment in response to the inducing cue, alkaline pH. Three of the six previously characterized cpxP loss-of-function mutations, M59T, Q55P, and Q128H, likely result from a destabilization of the protein fold, whereas the R60Q, D61E, and D61V mutations may alter intermolecular interactions.

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM–sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM’s N-terminal ribbon–helix–helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM.

TraM is essential for F plasmid‐mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 Å crystal structure of the TraM core tetramer (TraM58–127). TraM58–127 is a compact eight‐helical bundle, in which the N‐terminal helices from each protomer interact to form a central, parallel four‐stranded coiled‐coil, whereas each C‐terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen‐bonded arrangement within the central four‐helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.