Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action

Differences between the structures of bacterial, archaeal, and eukaryotic ribosomes account for the selective action of antibiotics. Even minor variations in the structure of ribosomes of different bacterial species may lead to idiosyncratic, species-specific interactions of the drugs with their targets. Although crystallographic structures of antibiotics bound to the peptidyl transferase center or the exit tunnel of archaeal (Haloarcula marismortui) and bacterial (Deinococcus radiodurans) large ribosomal subunits have been reported, it remains unclear whether the interactions of antibiotics with these ribosomes accurately reflect those with the ribosomes of pathogenic bacteria. Here we report X-ray crystal structures of the Escherichia coli ribosome in complexes with clinically important antibiotics of four major classes, including the macrolide erythromycin, the ketolide telithromycin, the lincosamide clindamycin, and a phenicol, chloramphenicol, at resolutions of ∼3.3 Å–3.4 Å. Binding modes of three of these antibiotics show important variations compared to the previously determined structures. Biochemical and structural evidence also indicates that interactions of telithromycin with the E. coli ribosome more closely resembles drug binding to ribosomes of bacterial pathogens. The present data further argue that the identity of nucleotides 752, 2609, and 2055 of 23S ribosomal RNA explain in part the spectrum and selectivity of antibiotic action.     

Sequence selectivity of macrolide-induced translational attenuation

The prevailing “plug-in-the-bottle” model suggests that macrolide antibiotics inhibit translation by binding inside the ribosome tunnel and indiscriminately arresting the elongation of every nascent polypeptide after the synthesis of six to eight amino acids. To test this model, we performed a genome-wide analysis of translation in azithromycin-treated Staphylococcus aureus. In contrast to earlier predictions, we found that the macrolide does not preferentially induce ribosome stalling near the 5′ end of mRNAs, but rather acts at specific stalling sites that are scattered throughout the entire coding region. These sites are highly enriched in prolines and charged residues and are strikingly similar to other ligand-independent ribosome stalling motifs. Interestingly, the addition of structurally related macrolides had dramatically different effects on stalling efficiency. Our data suggest that ribosome stalling can occur at a surprisingly large number of low-complexity motifs in a fashion that depends only on a few arrest-inducing residues and the presence of a small molecule inducer.During translation, nascent polypeptides travel through a ∼100-Å-long aqueous tunnel in the large ribosomal subunit before entering the cytoplasm. The ribosome tunnel is highly irregular in shape and contains numerous cavities and grooves (1). Despite its narrow diameter (∼15 Å on average), recent studies have indicated that at least some nascent chains adopt a more compact conformation (2–4). Furthermore, the detection of specific peptide sequence motifs can arrest elongation or termination in both eukaryotes and prokaryotes (5). The cis-acting translational attenuator serves to regulate the expression of downstream genes in response to physiological signals (6, 7) and small molecules, such as antibiotics or amino acids (8–10). In bacteria, ribosome stalling at the upstream element often promotes the rearrangement of structured mRNA and activates downstream translation. Many resistant pathogens exploit the translational stalling mechanism to up-regulate the synthesis of antibiotic resistance genes (10). Curiously, the ribosome-stalling peptides identified to date vary in length and share no sequence homology.The ribosome tunnel is the target of many clinically important macrolide antibiotics. Macrolides bind to the tunnel close to the peptidyltransferase center (PTC) and are thought to inhibit translation by physically obstructing the progression of the nascent chain, thereby leading to the premature drop-off of peptides that are only six to eight amino acids long (11). The prevailing view of macrolide action suggests that these antibiotics indiscriminately block the elongation of every protein during the early stage of translation. In contrast to this model, recent studies have shown that several proteins are able to bypass the inhibition, presumably by threading through the drug-bound tunnel or by stimulating dissociation of antibiotic from the ribosome (12–14). Although the precise mechanism is unclear, the identification of a short N-terminal motif that circumvents erythromycin blockage (12) suggests that the extent of macrolide inhibition is highly sequence-specific. The full scope of this selectivity remains unknown.To identify the protein sequence motifs that are susceptible and resistant to macrolide action, we performed ribosome profiling to examine the Staphylococcus aureus translatome in the presence of the macrolide azithromycin (AZ). In addition to identifying previously unidentified ORFs and noncoding RNAs, we found incomplete down-regulation of the translation in cells exposed to AZ. Of particular interest, we found that AZ-mediated translation arrest selectively occurs at stalling sites that are enriched in proline and charged residues, despite a lack of overall sequence conservation. Surprisingly, many ribosomes do not enrich the N-terminal region as predicted; instead, they undergo arrest at a much later stage of translational elongation depending on the locations of specific stalling sites. Even more unexpectedly, the AZ-bound ribosomes have the ability to overcome the initial AZ inhibition within a multistalling-site-containing sequence, possibly by rerouting the path of the nascent chain.     

The Upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed −1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed −1 frameshift efficiency. A upf3-Δ strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed −1 ribosomal frameshift efficiency resulting in loss of the M₁ virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.     

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.     

Mutant EGFR is required for maintenance of glioma growth in vivo,and its ablation leads to escape from receptor dependence

Epidermal growth factor receptor (EGFR) gene amplification is the most common genetic alteration in high-grade glioma, and ≈50% of EGFR-amplified tumors also harbor a constitutively active mutant form of the receptor, ΔEGFR. Although ΔEGFR greatly enhances tumor growth and is thus an attractive target for anti-glioma therapies, recent clinical experiences with EGFR kinase inhibitors have been disappointing, because resistance is common and tumors eventually recur. Interestingly, it has not been established whether ΔEGFR is required for maintenance of glioma growth in vivo, and, by extension, if it truly represents a rational therapeutic target. Here, we demonstrate that in vivo silencing of regulatable ΔEGFR with doxycycline attenuates glioma growth and, therefore, that it is crucial for maintenance of enhanced tumorigenicity. Similar to the clinical experience, tumors eventually regained aggressive growth after a period of stasis, but interestingly, without re-expression of ΔEGFR. To determine how tumors acquired this ability, we found that a unique gene, KLHDC8, herein referred to as SΔE (Substitute for ΔEGFR Expression)-1, is highly expressed in these tumors, which have escaped dependence on ΔEGFR. SΔE-1 is also expressed in human gliomas and knockdown of its expression in ΔEGFR-independent “escaper” tumors suppressed tumor growth. Taken together, we conclude that ΔEGFR is required for both glioma establishment and maintenance, and that gliomas undergo selective pressure in vivo to employ alternative compensatory pathways to maintain aggressiveness in the event of EGFR silencing. Such alternative pathways function as substitutes for ΔEGFR signaling and should therefore be considered as potential targets for additional therapy.     

The general mode of translation inhibition by macrolide antibiotics

Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. To understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5′-proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The problematic sequence motifs are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segment that contacts the antibiotic molecule in the exit tunnel. Therefore, it appears that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not functioning during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics.Macrolide antibiotics are among the most successful antibacterials and have been widely used for the treatment of serious infections. These drugs stop bacterial growth by inhibiting protein synthesis. Macrolides bind to the ribosome in the nascent peptide exit tunnel (NPET), a narrow conduit that the polypeptides assembled in the peptidyl transferase center (PTC) pass through on their way out of the ribosome (1–3). Binding of a macrolide molecule in the NPET at a short distance from the PTC obstructs the passage of the nascent peptides. Treatment of sensitive cells with macrolide antibiotics leads to a rapid decline of the overall protein synthesis and accumulation of peptidyl-tRNA (4). In the cell-free translation system, these drugs were shown to inhibit the synthesis of model polypeptides and cause early peptidyl-tRNA drop-off (5, 6). Based on these observations, it was largely assumed that macrolides indiscriminately inhibit the production of all cellular polypeptides by preventing the nascent chain egress and blocking translation at its early rounds (7).This general view, which has prevailed over decades, has been challenged by several recent findings. Crystallographic studies have shown that macrolides, although significantly narrowing the NPET, nevertheless still leave sufficient room for the growing nascent protein chain, thereby raising the possibility that some proteins may bypass the constriction created by the drug (3). Indeed, progression of the drug-bound ribosome along mRNAs can be halted in vitro at codons distal to the beginning of the ORF (8). Some biochemical evidence argues that the action of macrolide antibiotics depends on the properties of the polypeptide being synthesized by the drug-bound ribosome as revealed by differential inhibition of in vitro translation of certain model and natural mRNA templates (8–10). Consistent with these observations, the sites of macrolide-induced programmed ribosome stalling at the regulatory leader ORFs, which control expression of the macrolide resistance genes, are defined by the sequence of the encoded nascent peptide (11–15). Importantly, the protein-specific action of macrolides is readily observed in vivo, because treatment of sensitive cells with even very high concentrations of antibiotics allows the continued production of a subset of cellular polypeptides (8).Despite the growing evidence that macrolides may affect protein synthesis in a sequence-specific manner, the general mode of action of these antibiotics remains obscure. Therefore, to understand the key principles of inhibition of translation by macrolides, we carried out genome-wide ribosome profiling analysis in Escherichia coli cells treated with two types of antibiotics and monitored drug-induced changes in the translation of individual genes. Erythromycin (ERY), one of the drugs used in our study, represents the prototype macrolide antibiotic, whereas the second drug, telithromycin (TEL), belongs to the newest generation of macrolides, called ketolides (Fig. 1A). The results of the ribosome profiling experiments and in vitro biochemical testing allowed us to define some of the amino acid sequence motifs that are difficult to synthesize for drug-bound ribosomes and to propose a generalized model for the action of macrolide antibiotics, alleviating discrepancies that confounded the previous models.Open in a separate windowFig. 1.Gene-specific action of macrolide antibiotics. (A) Chemical structures of ERY and TEL antibiotics used in the profiling experiments. (B–D) Three common patterns of macrolide action observed in ribosome profiling analysis: (B) inhibition of translation at early stages, (C) translation arrest at the internal codons of the gene, and (D) translation through the entire length of the gene.     

Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases

Alterations of the epidermal growth factor receptor (EGFR) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2–7, resulting in truncation of the extracellular domain (ΔEGFR or EGFRvIII), which occurs in a large fraction of de novo malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-X_L. Here, we demonstrate that the ΔEGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-X_L was elevated in U87MG.ΔEGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.ΔEGFR cells. These responses were highly specific to constitutively kinase-active ΔEGFR, because overexpression of kinase-deficient ΔEGFR (DK) or wild-type EGFR had no such effects. Correspondingly, ΔEGFR specific tyrosine kinase inhibitors reduced Bcl-X_L expression and potentiated CDDP-induced apoptosis in U87MG.ΔEGFR cells. Ectopic overexpression of Bcl-X_L in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing ΔEGFR.     

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

The chloroplast NAD(P)H dehydrogenase (NDH) C (ndhC) and ndhK genes partially overlap and are cotranscribed in many plants. We previously reported that the tobacco ndhC/K genes are translationally coupled but produce NdhC and NdhK, subunits of the NDH complex, in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low. Hence, these findings suggested that the ndhK cistron is translated not only from the ndhC 5′UTR but also by an additional pathway. Using an in vitro translation system from tobacco chloroplasts, we report here that free ribosomes enter, with formylmethionyl-tRNA^fMet, at an internal AUG start codon that is located in frame in the middle of the upstream ndhC cistron, translate the 3′ half of the ndhC cistron, reach the ndhK start codon, and that, at that point, some ribosomes resume ndhK translation. We detected a peptide corresponding to a 57-amino-acid product encoded by the sequence from the internal AUG to the ndhC stop codon. We propose a model in which the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes to the ndhK cistron to produce NdhK in the amount required for the assembly of the NDH complex. This pathway is a unique type of translation to produce protein in the needed amount with the cost of peptide synthesis.     

Macrolide antibiotics allosterically predispose the ribosome for translation arrest

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.Expression of several bacterial and eukaryotic genes is controlled by nascent peptide-dependent programmed translation arrest. In the general scenario, ribosome stalling at an upstream regulatory ORF (uORF) triggers isomerization of the mRNA structure, leading to activation of expression of downstream cistron(s). Translation arrest ensues when a distinctive amino acid sequence (the “stalling domain”) of the growing chain assembled in the ribosomal peptidyl transferase center (PTC) is placed in the nascent peptide exit tunnel (NPET). Ribosome stalling may require additional signals, thereby making this gene control mechanism sensitive to the physiological state of the cell or to the chemical composition of the environment. Often the external signal is a small molecule whose binding to the ribosome renders translation responsive to specific nascent peptides (reviewed in refs. 1, 2). In most of the examined cases of cofactor- and nascent peptide-dependent translation arrest, the binding site of the cofactor in the ribosome is unknown, which hampers understanding of the interplay among the cofactor, the nascent peptide, and the ribosome. The exception is the inducible antibiotic resistance, in which ribosome stalling and gene activation rely on binding of an antibiotic to a well-defined site in the ribosome.Expression of macrolide resistance genes is triggered by drug-induced ribosome stalling at a defined codon of the uORF (3–5). Macrolides, from the prototype erythromycin (ERY) to the newest macrolide derivatives—ketolides, e.g., solithromycin (SOL)—bind in the NPET at a short distance from the PTC (6–9) (Fig. 1A). When a nascent peptide grows to 4–7 aa, it reaches the site of antibiotic binding and has to negotiate the drug-obstructed NPET aperture. Subsequent events depend on the properties of the nascent chain (3, 10, 11). Although for many proteins the encounter of the peptide with the antibiotic results in peptidyl–tRNA dropoff, the N-termini of certain nascent peptides can bypass the antibiotic. Translation of some of such proteins can be arrested at specific sites within the gene, resulting in formation of a stable stalled complex (11). Such translation arrest defines the role of macrolides as cofactors of programmed ribosome stalling (3, 10–12).Open in a separate windowFig. 1.Antibiotic and nascent peptide in the ribosomal exit tunnel. (A) The relative locations of the macrolide binding site in the NPET and the PTC active site were rendered by aligning crystallographic structures of Thermus thermophilus 70S ribosome complexed with aminoacylated donor and acceptor tRNA substrates [Protein Data Bank (PDB) ID codes 2WDK, 2WDL (25)] and the vacant ribosome complexed with ERY [PDB ID codes 3OHC, 3OHJ (9)]. The PTC active site, defined as the middistance between the attacking amino group of the acceptor substrate and the carbonyl carbon atom of the donor, is marked by an asterisk. (B) The modeled position of the 9-aa–long ErmCL nascent peptide in the ribosomal tunnel obstructed by ERY (19). In the stalled complex, ErmCL is juxtaposed with the antibiotic in the tunnel.The regulatory leader peptides of macrolide resistance genes have been classified by the structure of their known or presumed stalling domains (4, 5). Translation of ErmAL1 and ErmCL peptides is arrested after the ribosome has polymerized the 8-aa (ErmAL1) or 9-aa (ErmCL) long nascent chains that carry the C-terminal stalling domains Ile-Ala-Val-Val (IAVV) and Ile-Phe-Val-Ile (IFVI), respectively (12–14). The drug-bound ribosome stalls because it cannot catalyze transfer of the peptide from the P-site peptidyl–tRNA to the A-site aminoacyl–tRNA (12, 14). Importantly, although the N-terminal sequences of these peptides are not critical, the N-terminal segments are required for translation arrest (12). The conservation of the distance of the stalling domain from the N-terminus among peptides of these classes (4) corroborates the importance of the nascent chain length for the arrest. The 8–9-aa long ErmAL1 or ErmCL stalling peptides reach far into the NPET and must be juxtaposed with the antibiotic molecule in the NPET; such apposition has been suggested to play a key role in the mechanism of arrest (12) (Fig. 1B). This view agrees with the strict structural requirements of the macrolide cofactor in which removal or modification of the C3 cladinose abolishes stalling, possibly by disrupting drug–peptide interactions (15).The resistance leader peptides of the third major class have been studied to a much lesser extent (16–18). These peptides were grouped together based on the presence of the Arg-Leu-Arg (RLR) motif in their sequence (4) (Table S1), although the role of this motif in programmed arrest has not been verified. Intriguingly, in striking contrast to the IAVV and IFVI classes, the placement of the RLR motif within these peptides is highly variable (4).By analyzing translation arrest controlled by the RLR peptides, we discovered that the N-terminus is dispensable and macrolide antibiotic can block peptide bond formation and halt translation when the nascent chain is only 3-aa long and barely reaches the antibiotic in the NPET. Structural probing and molecular dynamics (MD) modeling showed the existence of an allosteric link between the NPET and the PTC, illuminating how binding of an antibiotic in the NPET predisposes the ribosome for stalling when translating specific amino acid sequences.     

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.     

Ricin inhibition of in vitro protein synthesis by plant ribosomes

In vitro translation systems were prepared with supernatant factors from wheat germ and 80S ribosomes from wheat germ, barley embryos, watermelon cotyledons, pea cotyledons, and castor bean endosperm. Ricin A-chain, which strongly inhibits protein synthesis by mammalian ribosomes, inhibited all of the plant ribosomal systems by 50% when present at 25-45 μg/ml—≈23,000 times the concentration needed to inhibit mammalian systems. Ricinus communis agglutinin A-chain, a protein similar to ricin A-chain, inhibited translation by the plant systems 50% at concentrations 5-10 times those of the ricin A-chain. Ribosomes from castor bean endosperm, the source of ricin and the agglutinin, were just as susceptible to the inhibitors as were ribosomes from the other four plants. Compartmentation of the inhibitors within vacuoles derived from protein bodies of the endosperm appears to be responsible for protecting cytoplasmic protein synthesis during germination of castor beans.     

Requirement for a membrane potential for cellulose synthesis in intact cells of Acetobacter xylinum

The marked lability in cell-free preparations of the enzyme system involved in cellulose biosynthesis in most organisms studied led us to investigate factors responsible for loss of activity on cellular disruption. Previous studies have led to the suggestion that the existence of a transmembrane electrical potential (ΔΨ) may be one factor responsible for maintaining an active system in intact cells. In this report, we show that dissipation of the ΔΨ in metabolizing cells of Acetobacter xylinum results in severe inhibition of cellulose synthesis. The effect can be reversed by restoration of the ΔΨ. Inhibition of cellulose biosynthesis by dissipation of the ΔΨ can be observed under conditions in which no substantial impairment of energy metabolism occurs—i.e., under conditions in which a transmembrane pH gradient is of sufficient magnitude to maintain an adequate overall protonmotive force across the membrane. The inhibition of cellulose biosynthesis is specifically related to changes in the ΔΨ, since the process can proceed normally in the absence of the pH gradient. These results support the suggestion that loss of the ΔΨ on cellular disruption may be one of the factors responsible for the low capacity for cellulose synthesis in isolated membrane preparations and also raise the possibility that modulation of the ΔΨ could be one means of regulating the rate of cellulose synthesis in vivo.     

In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state

It was shown decades ago that purified 30S ribosome subunits readily interconvert between “active” and “inactive” conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactive-to-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch.Forty-five years ago, Zamir, Elson, and their colleagues reported that purified 30S subunits of the ribosome undergo a readily reversible conformational change between “active” and “inactive” states and proposed that this conformational rearrangement might mimic a natural process (1). Noller and coworkers used chemical probing to show that this conformational change occurs in the neck and decoding center regions of the 16S ribosomal RNA (rRNA) and has “the appearance of a reciprocal interconversion between two differently structured states” (2). Recent structural analyses indicate that the protein-free 16S rRNA adopts alternative base-paired conformations in the neck region that are conserved among diverse eubacterial and archeal organisms (3). The ability to sample multiple conformations in this region is also conserved in eukaryotes (4). The original studies on the inactive and active states noted that probing ribosomes in cells might allow the biological roles of these states to be established (1, 2). Here we make use of recent innovations in in-cell RNA SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) probing (5) to interrogate the structure of 16S rRNA in free 30S subunits, in actively translating ribosomes, and in mutant ribosomes in exponentially growing Escherichia coli.     

A molecular mechanism underlying the neural-specific defect in torsinA mutant mice

A striking but poorly understood feature of many diseases is the unique involvement of neural tissue. One example is the CNS-specific disorder DYT1 dystonia, caused by a 3-bp deletion (“ΔE”) in the widely expressed gene TOR1A. Disease mutant knockin mice (Tor1a^ΔE/ΔE) exhibit disrupted nuclear membranes selectively in neurons, mimicking the tissue specificity of the human disease and providing a model system in which to dissect the mechanisms underlying neural selectivity. Our in vivo studies demonstrate that lamina-associated polypeptide 1 (LAP1) and torsinB function with torsinA to maintain normal nuclear membrane morphology. Moreover, we show that nonneuronal cells express dramatically higher levels of torsinB and that RNAi-mediated depletion of torsinB (but not other torsin family members) causes nuclear membrane abnormalities in Tor1a^ΔE/ΔE nonneuronal cells. The Tor1a^ΔE/ΔE neural selective phenotype therefore arises because high levels of torsinB protect nonneuronal cells from the consequences of torsinA dysfunction, demonstrating how tissue specificity may result from differential susceptibility of cell types to insults that disrupt ubiquitous biological pathways.     

The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling

The GPIbα-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIba^Δsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbα^Δsig/Δsig platelets bound von Willebrand factor (VWF) normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GPIbα^Δsig/Δsig platelets with ADP and thrombin was normal, but GPIbα^Δsig/Δsig platelets stimulated with collagenrelated- peptide (CRP) exhibited markedly decreased P-selectin exposure and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in GPVI-mediated signaling. Consistent with this, while hemostasis was normal in GPIbα^Δsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GPIbα^Δsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GPIbα^Δsig/Δsig platelets but to a lesser extent than those with CRP. GPIbα^Δsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation contributes to the phenotype of the GPIbα^Δsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.     

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na⁺ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na⁺] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na⁺ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.     

Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations

Gram-negative bacteria have an outer membrane containing LPS. LPS is constituted of an oligosaccharide portion and a lipid-A moiety that embeds this molecule within the outer membrane. LPS is a pathogen-associated molecular pattern, and several pathogens modify their lipid-A as a stealth strategy to avoid recognition by the innate immune system and gain resistance to host factors that disrupt the bacterial cell envelope. An essential feature of Salmonella enterica Typhimurium pathogenesis is its ability to replicate within vacuoles in professional macrophages. S. Typhimurium modifies its lipid-A by hydroxylation by the Fe2+/α-ketoglutarate-dependent dioxygenase enzyme (LpxO). Here, we show that a periplasmic protein of the bacterial oligonucleotide/oligosaccharide-binding fold family, herein named virulence and stress-related periplasmic protein (VisP), on binding to the sugar moiety of peptidoglycan interacts with LpxO. This interaction inhibits LpxO function, leading to decreased LpxO-dependent lipid-A modifications and increasing resistance to stressors within the vacuole environment during intramacrophage replication promoting systemic disease. Consequently, ΔvisP is avirulent in systemic murine infections, where VisP acts through LpxO. Several Gram-negative pathogens harbor both VisP and LpxO, suggesting that this VisP-LpxO mechanism of lipid-A modifications has broader implications in bacterial pathogenesis. Bacterial species devoid of LpxO (e.g., Escherichia coli) have no lipid-A phenotypes associated with the lack of VisP; however, VisP also controls LpxO-independent phenotypes. VisP and LpxO act independently in the S. Typhimurium murine colitis model, with both mutants being attenuated for diverging reasons; ΔvisP is less resistant to cationic antimicrobial peptides, whereas ΔlpxO is deficient for epithelial cell invasion. VisP converges bacterial cell wall homeostasis, stress responses, and pathogenicity.     

Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation

We present an approach for monitoring protein–protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing β-galactosidase (β-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the β-gal activity resulting from the forced interaction of nonfunctional weakly complementing β-gal peptides (Δα and Δω) serves as a measure of the extent of interaction of the non-β-gal portions of the chimeras. To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Δα and Δω, respectively. Enzymatic β-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.     

Cytoplasmic and chloroplast synthesis of phycobilisome polypeptides

In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the α and β subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the α and β subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.     

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3′–5′ exonuclease proofreading function

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD′₂C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3′–5′ exonuclease proofreading activity of the pol III -subunit also is disabled. TR was measured in isogenic uvrA6 ΔumuDC strains carrying the dominant negative dnaQ allele, mutD5, or ΔdnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T–T dimer. As expected, little TR was observed in the ΔumuDC dnaQ⁺ strain. Surprisingly, 26% TR occurred in UV-irradiated ΔumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC⁺mutD5 strain. lexA3 (Ind⁻) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for ≈70% of the TR, and another LexA-independent, responsible for the remaining ≈30%. Curiously, the ΔumuDC ΔdnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in ΔdnaQ strains, since introduction of spq-2 into the ΔumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041–6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the ΔumuDC mutD5 strain is unchanged by introduction of a ΔpolB mutation.