Insights into ParB spreading from the complex structure of Spo0J and parS

Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein–protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.The integrity of chromosomes and plasmids relies on precise DNA replication and segregation (1, 2). The initiation of DNA replication has to synchronize with the cell cycle to ensure precise chromosome segregation (3). In bacteria, the chromosome-encoded plasmid-partitioning system (Par) (4) and the structural maintenance of chromosomes (SMC) condensation complex (5) are two highly conserved systems associate with chromosome segregation and organization. SMC contributes to the overall stability and organization of genome (6–8). The partition system denoted ParABS is comprised of two proteins (ParA and ParB) and a centromere-like DNA element (parS) (9). ParB binds specifically to parS to form a complex. After binding ATP, ParA can interact with the ParB–parS complex to form a nucleoid–adaptor complex. ParB promotes the ATP hydrolysis activity of the complex to separate the chromosomes (9–13).In the bacterial chromosomal ParABS system, ParB has two functions: one is to regulate chromosome replication and sporulation (8, 12, 14) and the other is to participate in chromosome segregation (5, 15–17). ParB spreads along the chromosomal DNA by binding at specific parS and nonspecific DNA sites to form a high-order partition complex (18–20). This partition complex is required for the loading of SMC onto the chromosomal DNA (5). In addition, the N-terminal domain of ParB can interact with ParA and stimulate its ATPase activity (21). This nucleoid–adaptor complex, ParA–ParB–parS is used to drive chromosome segregation (22, 23). However, the detailed mechanism for this process is still unclear.Members of the ParB superfamily share similar functional domains: an N-terminal domain for protein–protein interactions, a central DNA-binding domain for parS binding, and a C-terminal domain for ParB dimerization (24). Two conserved N-terminal domain residues, Lys3 and Lys7, in the ParB from Bacillus subtilis (BsSpo0J), have been shown to interact with its regulatory protein BsSoj, a member of the ParA superfamily (3). The loss-of-function BsSpo0J R80A mutant was originally discovered by Autret et al. (25) and reportedly has disrupted focus formation by fluorescence microscopy. More recently, Graham et al. (20) showed that BsSpo0J bridges chromosomal DNA using single-molecule experiments. However, its R79A, R80A, and R82A mutants could not spread in vivo and did not bridge DNA in vitro. These highly conserved arginine residues were defined as an arginine patch (20). Furthermore, Broedersz et al. (26) studied the condensation and localization of ParB by computational simulation.The crystal structures of ParB superfamily proteins have been reported for a DNA-free form of TtSpo0J (from Thermus thermophilus, containing the N-terminal and the DNA-binding domains) (10) and three complexes: the RP4–KorB-O_B complex (from plasmid RP4, containing the DNA-binding domain) (27), the P1 ParB–parS complex (from Enterobacteria phage P1, containing the DNA-binding and the C-terminal domains) (28), and the F-SopB–sopC complex (from plasmid F, containing the DNA-binding domain) (29).The Helicobacter pylori ParABS system consists of HpSoj (ParA), HpSpo0J (ParB), and parS DNA (30, 31). Herein, we report the crystal structure of a C-terminal domain truncated HpSpo0J (Ct-HpSpo0J)–parS complex. The N-terminal and the DNA-binding domains are present on Ct-HpSpo0J. The structural details of the complex in combination with results from EMSAs, fluorescence anisotropy assay, and small angle X-ray scattering (SAXS) allow us to propose a model for ParB spreading as it relates to chromosome segregation.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.Proper protein biogenesis is a prerequisite for the maintenance of a functional proteome. Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1–5) such as trigger factor (TF) (1, 4, 6, 7), Hsp70, and the nascent polypeptide-associated complex (8–13); modification enzymes (10, 14–16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17–20), SecA (21), the SecYEG (or Sec61p) (22, 23) and YidC translocases (24, 25), and the ribosome-bound quality control complex (26–30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control. Within seconds after the nascent polypeptide emerges from the ribosomal exit tunnel, it must engage the correct set of factors and thus commit to the proper biogenesis pathway. How this is accomplished in the crowded environment at the ribosome exit site is an emerging question. In this work, we address this question by deciphering how nascent proteins are selected between two major protein biogenesis machineries in bacteria, SRP and TF.SRP is a universally conserved ribonucleoprotein complex responsible for the cotranslational targeting of proteins to the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane (31). SRP recognizes ribosome-nascent chain complexes (termed RNC or cargo) carrying strong signal sequences and delivers them to the SecYEG or YidC translocation machinery on the target membrane. SRP binds RNC via two interactions: a helical N domain in the SRP54 protein (called Ffh in bacteria) binds the ribosomal protein L23, and a methionine-rich M domain binds hydrophobic signal sequences on nascent proteins as they emerge from the translating ribosome (Fig. 1A). Both SRP and SRP receptor (called FtsY in bacteria) also contain a conserved NG domain, comprised of a GTPase (guanosine 5′-triphosphate hydrolase) G domain and the N domain, whose direct interaction mediates the delivery of cargo to the target membrane.Open in a separate windowFig. 1.TF binds to SRP-occupied RNCs and weakens SRP binding. (A) Schematic depiction of the FRET assay to measure RNC–SRP binding. Green dot denotes Cm (donor); red dot denotes BODIPY FL (acceptor). (B) N-terminal sequences of the different substrates used in this study. Bold highlights the hydrophobic core of the signal sequences. Asterisk denotes the position where the amino acid is replaced by the Cm dye. (C and D) Equilibrium titrations for RNC–SRP binding in the presence of increasing TF concentration (indicated as increasing shades of red). The data were fitted to Eq. S2 and yielded the following parameters. (C) Apparent K_d values for RNC_FtsQ binding of 1.1 nM, 1.5 nM, 9.2 nM, and 16.6 nM and FRET end points of 0.54, 0.35, 0.29, and 0.17, respectively, with 0 µM, 1 µM, 5 µM, and 30 µM TF present. (D) Apparent K_d values for RNC_phoA binding of 17.2 nM, 21.1 nM, 30.3 nM, 28.3 nM, 31.5 nM, 104.5 nM, 106.3 nM, and 131.9 nM and FRET end points of 0.40, 0.41, 0.39, 0.29, 0.21, 0.19, 0.09, and 0.08, respectively, with 0 µM, 0.1 µM, 0.2 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, and 10 µM TF present. (E) Summary of the effect of TF on apparent RNC–SRP binding affinity with the different substrates. The red dashed line denotes the cellular SRP concentration. Error bars are shown but may not be visible. Error bars are SDs from two to three measurements.Biophysical analyses (32–34) showed that membrane targeting is a two-step process in which SRP and FtsY first associate via their N domains to form a transient early intermediate (31, 32, 35). GTP (guanosine 5′-triphosphate)-driven rearrangements then bring the G domains of both proteins into close contact, giving a stable closed complex (36, 37). This rearrangement also exposes a membrane-binding helix of FtsY and thus is coupled to the membrane targeting of cargo (38). Importantly, SRP•FtsY assembly contributes extensively to the fidelity of SRP (39). The initial recognition of RNC by SRP is insufficient to reject suboptimal cargos bearing weak signal sequences (40, 41). Instead, a correct cargo strongly stabilizes the otherwise labile early intermediate and thus accelerates formation of the SRP•FtsY closed complex over 10³-fold, whereas suboptimal cargos provide much less stimulation (34, 40, 42). This enables rapid delivery of the correct cargos to the target membrane and provides kinetic discrimination against suboptimal cargos (Fig. S1).TF is a major cotranslational chaperone in bacteria, with an estimated cellular concentration of 50–80 µM (6). With a dissociation constant (K_d) of ∼1 µM for ribosomes (43), TF is bound to virtually every ribosome in the cell. Like SRP, TF contacts the ribosome via the L23 and L29 proteins near the ribosome exit site (3, 5, 44). Also analogous to SRP, TF preferentially interacts with hydrophobic sequences on the nascent polypeptide (1, 2, 4, 45, 46), mediated by a large concave surface rich in hydrophobic residues (1, 3–6). Despite these similarities with SRP, TF directs substrate proteins to distinct biogenesis pathways: It exhibits synthetic lethality with DnaK/J and facilitates the productive folding of cytosolic proteins (1, 4, 7, 9, 11). It also interacts with a subset of secretory and outer membrane proteins and interfaces with the posttranslational SecA/B pathway (8, 10, 12–14).SRP and TF are two distinct biogenesis pathways that a nascent protein must commit to. This raises intriguing questions: How do these two factors, which have overlapping substrate preferences, compete and/or collaborate at the ribosome exit site? How are nascent proteins sorted between them and committed to the correct pathway in a timely and accurate manner? Extensive past work to address these questions has led to different (and sometimes contradictory) models, including (i) TF and SRP compete for binding to the RNC (10, 15, 16, 18); (ii) TF and SRP can bind to the same RNC simultaneously (17, 19–21); (iii) FtsY rejects TF from SRP-bound ribosomes (17); and (iv) TF preferentially occupies longer nascent chains (13, 45–47) and, by inference, SRP preferentially binds short nascent chains. A unifying model that reconciles all these observations and explains how nascent chains on the ribosome are selected by TF or SRP is still lacking. Most importantly, most of the previous studies have focused on the initial binding of SRP or TF to the nascent polypeptide, which may not be the step at which nascent proteins are committed to their respective biogenesis pathways.In this work, we used high-resolution biochemical and biophysical analyses to investigate the interplay between TF and SRP at the ribosome exit site in molecular detail. We show that TF regulates SRP function by three distinct mechanisms, which together enhance the ability of the SRP pathway to reject suboptimal substrates. Our results establish a comprehensive and cohesive model that explains previous observations, delineates the complex interplay between protein biogenesis factors at the ribosome exit site, and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved in this crowded environment.     

Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-? resolution

Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16-Å resolution structures of RPPs bound to vacant or nontranslating ribosomes. Here we present a cryo-electron microscopy reconstruction of the RPP TetM in complex with a translating ribosome at 3.9-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM with a unique splayed conformation of nucleotides A1492 and A1493 at the decoding center of the small subunit. The resolution enables us to unambiguously model the side chains of the amino acid residues comprising loop III in domain IV of TetM, revealing that the tyrosine residues Y506 and Y507 are not responsible for drug-release as suggested previously but rather for intrafactor contacts that appear to stabilize the conformation of loop III. Instead, Pro509 at the tip of loop III is located directly within the tetracycline binding site where it interacts with nucleotide C1054 of the 16S rRNA, such that RPP action uses Pro509, rather than Y506/Y507, to directly dislodge and release tetracycline from the ribosome.The ribosome is one of the major targets for antibiotics within the bacterial cell (1, 2). A well-characterized class of broad-spectrum antibiotics in clinical use are the tetracyclines, which bind to elongating ribosomes and inhibit delivery of the EF-Tu•GTP•aa-tRNA ternary complex to the A-site (1, 3). X-ray crystal structures of ribosomal particles in complex with tetracycline have revealed that the primary drug binding site is located in helix 34 (h34) of the 16S rRNA, overlapping the binding position of the anticodon-stem loop of an A-site tRNA (4–6). The widespread use of tetracyclines has led to an increase in tetracycline resistance among clinically relevant pathogenic bacteria, thus limiting the medical utility of many members of this class (7). Drug efflux and ribosome protection are the most common tetracycline resistance mechanisms acquired by bacteria (8) and have led to the development of the third generation of tetracycline derivatives, such as tigecycline, which display enhanced antimicrobial activity and overcome both the efflux and ribosome protection resistance mechanisms (6, 9–11).To date, there are 12 distinct classes of ribosome protection proteins (RPPs) that confer resistance to tetracycline, with the most prevalent and best characterized being TetO and TetM (3, 8, 12). The different classes of RPPs have high homology with one another; for example, Campylobacter jejuni TetO displays >75% identity (>85% similarity) with Enterococcus faecalis TetM. Based on the presence of conserved nucleotide binding motifs, RPPs are grouped together within the translation factor superfamily of GTPases (13). Accordingly, TetO and TetM catalyze the release of tetracycline from the ribosome in a GTP-dependent manner (14, 15). Biochemical studies indicate that, although GTPase activity is necessary for multiturnover of RPPs, GTP hydrolysis is not strictly required to dislodge tetracycline because the drug is also released when nonhydrolysable GTP analogs are used (14, 15).Nonhydrolysable GTP analogs have been used to trap RPPs on the ribosome for structural analysis by cryo-EM. The first structure of an RPP-ribosome complex was a cryo-EM reconstruction of a TetO•70S complex at 16-Å resolution. This structure revealed that TetO binds analogously to the ribosome as translation elongation factor EF-G (16), consistent with the significant homology (∼25/35% identity/similarity) between RPPs and EF-G (17). Because the electron density for TetO did not come within 6 Å of the tetracycline-binding site (16), TetO was suggested to chase the drug from the ribosome by inducing conformational changes within h34 (12, 16, 18). In contrast, two subsequent structures at higher resolution, a TetM•70S complex at 7.2 Å (19) and a TetO•70S complex at 9.6 Å (20), revealed electron density for the RPPs directly overlapping with the tetracycline binding site. Based on the homology with EF-G, molecular models for the RPPs were generated and docked into the cryo-EM maps, suggesting that residues within loop III of domain IV of TetM/TetO come into direct contact with the tetracycline molecule (19, 20). Consistently, mutagenesis studies identified specific residues within loop III that are critical for RPP activity (19–21), in particular the conserved tyrosine residues Y506 and Y507 (19, 20). However, the exact role of these tyrosine residues and a detailed molecular understanding of the mechanism by which RPPs dislodge tetracycline from its binding site was not possible at the reported resolutions.Here we present a cryo-EM structure of TetM in complex with a translating ribosome at an average resolution of 3.9 Å. Local resolution calculations indicate that the majority of the core of the ribosome and domain IV of TetM extends toward 3.5 Å, enabling bulky side chains to be modeled. We provide a detailed account of the interactions between TetM and the ribosome, in particular revealing a complex network of interactions of the C-terminal helix and domain IV of TetM with the ribosomal decoding site and intersubunit bridge B2a. The structure reveals that Pro509 at the tip of loop III, rather than the previously identified tyrosine Y506 and Y507, overlaps the binding site of tetracycline and is therefore directly involved in releasing tetracycline from the ribosome.     

Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation

During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.During protein synthesis the ribosome iteratively incorporates new amino acids delivered by aminoacylated transfer RNAs (tRNA) into the growing polypeptide chain in a manner specified by the codons in a messenger RNAs (mRNA). This elongation cycle is controlled by the two translocational GTPases elongation factors (EF)-Tu and EF-G. Following EF-Tu–dependent delivery of aminoacyl-tRNA to the A site and peptide bond formation, the ribosome adopts a pretranslocational state containing a peptidyl A-site tRNA and a deacylated P-site tRNA. In the subsequent translocation reaction, the interplay between the ribosome and elongation factor EF-G shifts the tRNAs from the A and P sites to the P and E sites, respectively. In each of these binding sites a tRNA contacts both ribosomal subunits and interacts with the 30S and 50S subunits via its anticodon-stem loop (ASL) and acceptor arm, respectively (1). Partial tRNA movement can occur before the EF-G–dependent translocation step, involving spontaneous and reversible movement of the tRNA acceptor arms relative to the large ribosomal subunit, which leads to a shift from classic A/A and P/P binding states into intersubunit A/P and P/E hybrid states (where the first and second letters indicate tRNA contacts on the small and large subunits, respectively) (2–4).A remarkable feature of translocation is the precise coupling of movement of the tRNAs together with the bound mRNA (designated as the tRNA₂•mRNA module), so that the mRNA advances by exactly one codon on the ribosome. Translocation is associated with large-scale conformational changes within the ribosomal complex, which includes rotation and back-rotation between the two subunits (during which the small subunit rotates counterclockwise and clockwise relative to the large subunit, viewing the ribosome from the solvent side of the small subunit) (5–7), and an additional forward and reverse swiveling movement of the 30S head (an autonomous domain of the 30S subunit, which can rotate around an axis roughly orthogonal to the axis of intersubunit rotation) (6, 8–13). Structural studies have suggested that intersubunit rotation within the pretranslocational complex is coupled to tRNA hybrid state formation (14–18). EF-G–dependent movement of the tRNAs and mRNA on the 30S subunit then occurs during reversal of the intersubunit rotation (6, 7). Moreover, multiparticle cryo-EM and X-ray crystallography studies suggest that movement of the tRNAs relative to the 30S subunit occurs via additional intermediate tRNA binding states, which are formed upon the back rotation of the 30S-body/platform and a large swiveling movement of the 30S head (6, 10). One of the first implications of swiveling of the 30S subunit head came from the studies of Schuwirth et al. (19), who observed a constriction of 13 Å between head and body of the 30S subunit that would block passage of tRNA between the P and E sites. These authors suggested that rotation of the 30S head would allow movement of the tRNA ASL, and could correspond to an unlocking event during translocation. Although swiveling of the small subunit head has been observed in different ribosomal complexes with bound EF-G (or eEF2) (6, 8–13) or with bound tRNAs (18), it has not been observed directly in the context of an authentic translocation complex containing EF-G together with two tRNAs. Previous structural analysis of translocation used model complexes where EF-G was directly bound to either vacant ribosomes, to ribosomal complexes with one tRNA, or to complexes in the posttranslocational state (5, 6, 8, 10–13, 20–22). The present study describes a cryo-EM reconstruction more closely resembling an authentic translocation intermediate, in which EF-G•GTP was bound to a canonical pretranslocational ribosomal complex containing two tRNAs and mRNA, and stalled during the translocation reaction by the antibiotic fusidic acid (FA). The resulting sample was analyzed by means of multiparticle cryo-EM (23). The classification yielded only a single major population of 70S•EF-G•GDP•FA particles trapped in an intermediate state of the translocation reaction. In contrast to all previously described 70S•EF-G structures (5, 6, 10–13, 20–22), the resulting reconstruction directly visualizes two tRNAs bound to the ribosome in two different chimeric intrasubunit hybrid states. The data presented here show how reciprocal conformational changes within the ribosome coordinate the synchronous movement of the mRNA and bound tRNA pair.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.