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Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control., (© 2023. The Author(s).)

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus , including Aspergillus fumigatus , the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans ( An UGP). To understand the molecular basis of An UGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native An UGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. An UGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for An UGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens., Competing Interests: The authors declare no conflict of interest.

There are multiple RNA degradation mechanisms in eukaryotes, key among these is mRNA decapping, which requires the Dcp1-Dcp2 complex. Decapping is involved in various processes including nonsense-mediated decay (NMD), a process by which aberrant transcripts with a premature termination codon are targeted for translational repression and rapid decay. NMD is ubiquitous throughout eukaryotes and the key factors involved are highly conserved, although many differences have evolved. We investigated the role of Aspergillus nidulans decapping factors in NMD and found that they are not required, unlike Saccharomyces cerevisiae. Intriguingly, we also observed that the disruption of one of the decapping factors, Dcp1, leads to an aberrant ribosome profile. Importantly this was not shared by mutations disrupting Dcp2, the catalytic component of the decapping complex. The aberrant profile is associated with the accumulation of a high proportion of 25S rRNA degradation intermediates. We identified the location of three rRNA cleavage sites and show that a mutation targeted to disrupt the catalytic domain of Dcp2 partially suppresses the aberrant profile of Δdcp1 strains. This suggests that in the absence of Dcp1, cleaved ribosomal components accumulate and Dcp2 may be directly involved in mediating these cleavage events. We discuss the implications of this., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Two years after its emergence, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several vaccines. The extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin converting enzyme 2 (ACE2) through its receptor binding domain (RBD), is the major target of neutralizing antibodies. Like to many other viral fusion proteins, the SARS-CoV-2 spike protein utilizes a glycan shield to thwart the host immune response. To grasp the influence of chemical signatures on carbohydrate mobility and reconcile the cryo-EM density of specific glycans we combined our cryo-EM map of the S ectodomain to 4.1 Å resolution, reconstructed from a limited number of particles, and all-atom molecular dynamics simulations. Chemical modifications modeled on representative glycans (defucosylation, sialylation and addition of terminal LacNAc units) show no significant influence on either protein shielding or glycan flexibility. By estimating at selected sites the local correlation between the full density map and atomic model-based maps derived from molecular dynamics simulations, we provide insight into the geometries of the α-Man-(1→3)-[α-Man-(1→6)-]-β-Man-(1→4)-β-GlcNAc(1→4)-β-GlcNAc core common to all N -glycosylation sites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stagnoli, Peccati, Connell, Martinez-Castillo, Charro, Millet, Bruzzone, Palazon, Ardá, Jiménez-Barbero, Ereño-Orbea, Abrescia and Jiménez-Osés.)

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30 S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16 S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1 H, 15 N, and 13 C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Omadacycline is an aminomethylcycline antibiotic with potent activity against many Gram-positive and Gram-negative pathogens, including strains carrying the major efflux and ribosome protection resistance determinants. This makes it a promising candidate for therapy of severe infectious diseases. Omadacycline inhibits bacterial protein biosynthesis and competes with tetracycline for binding to the ribosome. Its interactions with the 70S ribosome were, therefore, analyzed in great detail and compared with tigecycline and tetracycline. All three antibiotics are inhibited by mutations in the 16S rRNA that mediate resistance to tetracycline in Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma hominis, and Propionibacterium acnes. Chemical probing with dimethyl sulfate and Fenton cleavage with iron(II)-complexes of the tetracycline derivatives revealed that each antibiotic interacts in an idiosyncratic manner with the ribosome. X-ray crystallography had previously revealed one primary binding site for tetracycline on the ribosome and up to five secondary sites. All tetracyclines analyzed here interact with the primary site and tetracycline also with two secondary sites. In addition, each derivative displays a unique set of non-specific interactions with the 16S rRNA., Competing Interests: This study was funded by Novartis. J.N.S. is an employee of Paratek Pharmaceuticals. Neither company had a role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear (13)C and (15)N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G-ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit 'pe/E' hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the 'missing link' in terms of tRNA intermediates involved in the universally conserved translocation process.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 microg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 microg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 microg/ml) were higher than those previously reported in C. jejuni (64 to 128 microg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 microg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 microg/ml for Escherichia coli DH5alpha. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 microg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 microg/ml) were identical to one other, although the tet(O) genes encoding a 512-microg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3'-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.

BipA is a highly conserved protein with global regulatory properties in Escherichia coli. We show here that it functions as a translation factor that is required specifically for the expression of the transcriptional modulator Fis. BipA binds to ribosomes at a site that coincides with that of elongation factor G and has a GTPase activity that is sensitive to high GDP:GTP ratios and stimulated by 70S ribosomes programmed with mRNA and aminoacylated tRNAs. The growth rate-dependent induction of BipA allows the efficient expression of Fis, thereby modulating a range of downstream processes, including DNA metabolism and type III secretion. We propose a model in which BipA destabilizes unusually strong interactions between the 5' untranslated region of fis mRNA and the ribosome. Since BipA spans phylogenetic domains, transcript-selective translational control for the 'fast-track' expression of specific mRNAs may have wider significance.

15N-(1)H NMR spectroscopy has been used to probe the dynamic properties of uniformly (15)N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex ( approximately 2.3 MDa), [(1)H-(15)N] heteronuclear single-quantum correlation spectra contain approximately 100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7/L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T(1)) and T(1 rho) (15)N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7/L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7/L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7/L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis.

In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes.

Tet(O) is an elongation factor-like protein which confers resistance to the protein synthesis inhibitor tetracycline by promoting the release of the drug from its inhibitory site on the ribosome. Here we investigated the interaction of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS) probing and binding assays, that it interacts preferentially with the post-translocational ribosome. Furthermore, using an XTP-dependent mutant of Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within the ribosome which can be detected by EF-Tu, and manifested as a stimulation in the GTPase activity of this elongation factor. As such, these conformational changes probably involve the ribosomal GTPase-associated center and, accordingly, Tet(O) alters the DMS modification pattern of the L11 region. Additionally, tetracycline binding is associated with an E(a) of 58 kJ/mol. These results suggest a model where both Tet(O) and tetracycline induce a conformational change in functionally opposite directions and the Tet(O)-induced conformation persists after it has left the ribosome; this prevents rebinding of the drug while allowing productive A-site occupation by a ternary complex in the presence of tetracycline.

Tet(o) is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet(o) interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet(o) changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet(o) binding site visualized by cryo-EM, indicating that Tet(o) induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet(o) unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(o). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis.

Our understanding of the process of translation has progressed rapidly since the availability of highly resolved structures for the ribosome. A wealth of information has emerged in terms of both RNA and protein structure and the interplay between them. This has prompted us to revisit the astonishing "treasure trove" of functional data regarding the ribosome that has accumulated over the past decades. Here we try a systematic synopsis of these ribosomal functions in light of the cryo-electron microscopic structures (resolution >7 A) and the atomic x-ray structures (>2.4 A) of the ribosome.

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.