Lamotrigine compromises the fidelity of initiator tRNA recruitment to the ribosomal P-site by IF2 and the RbfA release from 30S ribosomes in Escherichia coli.

Lamotrigine (Ltg), an anticonvulsant drug, targets initiation factor 2 (IF2), compromises ribosome biogenesis and causes toxicity to Escherichia coli. However, our understanding of Ltg toxicity in E. coli remains unclear. While our in vitro assays reveal no effects of Ltg on the ribosome-dependent GTPase activity of IF2 or its role in initiation as measured by dipeptide formation in a fast kinetics assay, the in vivo experiments show that Ltg causes accumulation of the 17S precursor of 16S rRNA and leads to a decrease in polysome levels in E. coli. IF2 overexpression in E. coli increases Ltg toxicity. However, the overexpression of initiator tRNA (i-tRNA) protects it from the Ltg toxicity. The depletion of i-tRNA or overexpression of its 3GC mutant (lacking the characteristic 3GC base pairs in anticodon stem) enhances Ltg toxicity, and this enhancement in toxicity is synthetic with IF2 overexpression. The Ltg treatment itself causes a detectable increase in IF2 levels in E. coli and allows initiation with an elongator tRNA, suggesting compromise in the fidelity/specificity of IF2 function. Also, Ltg causes increased accumulation of ribosome-binding factor A (RbfA) on 30S ribosomal subunit. Based on our genetic and biochemical investigations, we show that Ltg compromises the function of i-tRNA/IF2 complex in ribosome maturation.

Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome.

Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3'-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.

Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of Giardia intestinalis.

Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-electron microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal proteins, but retain some bacterial features. The translocation intermediates, with naturally bound tRNAs and eukaryotic elongation factor 2 (eEF2), display characteristic ribosomal intersubunit rotation and small subunit's head swiveling-universal for translocation. In addition, we observe the eukaryote-specific 'subunit rolling' dynamics, albeit with limited features. Finally, the eEF2·GDP state features a uniquely positioned 'leaving phosphate (Pi)' that proposes hitherto unknown molecular events of Pi and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes from both the structural and mechanistic perspectives.

Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria.

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

p53 Isoforms as Cancer Biomarkers and Therapeutic Targets.

This review aims to summarize the implications of the major isoforms of the tumor suppressor protein p53 in aggressive cancer development. The current knowledge of p53 isoforms, their involvement in cell-signaling pathways, and their interactions with other cellular proteins or factors suggests the existence of an intricate molecular network that regulates their oncogenic function. Moreover, existing literature about the involvement of the p53 isoforms in various cancers leads to the proposition of therapeutic solutions by altering the cellular levels of the p53 isoforms. This review thus summarizes how the major p53 isoforms Δ40p53α/ß/γ, Δ133p53α/ß/γ, and Δ160p53α/ß/γ might have clinical relevance in the diagnosis and effective treatments of cancer.

Repurposing tRNAs for nonsense suppression.

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

Mechanistic insights into translation inhibition by aminoglycoside antibiotic arbekacin.

How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.

Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis.

The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexes mediate cotranslational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of unchaperoned protein in the aqueous phase. Here, we used giant unilamellar vesicles encapsulating minimal translation components to systematically interrogate the requirements for insertion of the model protein proteorhodopsin (PR) - a structurally ubiquitous membrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for cotranslational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a "pretranslocon" era, and they offer new opportunities for generating artificial cells.

Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.

Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.

GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors.

Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a "compact to open" conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the "compact to open" transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo.

Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from "Generalist" Ancestors.

It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, "generalist" ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.

Real-time measurements of aminoglycoside effects on protein synthesis in live cells.

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Ribosomal RNA Modulates Aggregation of the Podospora Prion Protein HET-s.

The role of the nucleic acids in prion aggregation/disaggregation is becoming more and more evident. Here, using HET-s prion from fungi Podospora anserina (P. anserina) as a model system, we studied the role of RNA, particularly of different domains of the ribosomal RNA (rRNA), in its aggregation process. Our results using Rayleigh light scattering, Thioflavin T (ThT) binding, transmission electron microscopy (TEM) and cross-seeding assay show that rRNA, in particular the domain V of the major rRNA from the large subunit of the ribosome, substantially prevents insoluble amyloid and amorphous aggregation of the HET-s prion in a concentration-dependent manner. Instead, it facilitates the formation of the soluble oligomeric "seeds", which are capable of promoting de novo HET-s aggregation. The sites of interactions of the HET-s prion protein on domain V rRNA were identified by primer extension analysis followed by UV-crosslinking, which overlap with the sites previously identified for the protein-folding activity of the ribosome (PFAR). This study clarifies a missing link between the rRNA-based PFAR and the mode of propagation of the fungal prions.

Selective translation by alternative bacterial ribosomes.

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation.

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.

Collateral Toxicity Limits the Evolution of Bacterial Release Factor 2 toward Total Omnipotence.

When new genes evolve through modification of existing genes, there are often tradeoffs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near-cognate stop codons and highlights a functional tradeoff that we term "collateral toxicity." We suggest that this type of tradeoff may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary tradeoffs between "old" and "new" functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds to alterations in the demand not only for RF2 activity but also for RF1 activity.

Author Correction: Inhibition of translation termination by small molecules targeting ribosomal release factors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibition of translation termination by small molecules targeting ribosomal release factors.

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

RNA modulates aggregation of the recombinant mammalian prion protein by direct interaction.

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general.

The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation.

Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.