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Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNA Gly and tRNA Thr . The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel SJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Toxic small alarmone synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin and secretion systems. toxSASs act through either translation inhibition mediated by pyrophosphorylation of transfer RNA (tRNA) CCA ends or synthesis of the toxic alarmone adenosine pentaphosphate ((pp)pApp) and adenosine triphosphate (ATP) depletion, exemplified by FaRel2 and FaRel, respectively. However, structural bases of toxSAS neutralization are missing. Here we show that the pseudo-Zn 2+ finger domain (pZFD) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor-binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable for FaRel neutralization. Collectively, our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Summary: Upstream open reading frames (uORFs, often encoding so-called leader peptides) can regulate translation and transcription of downstream main ORFs (mORFs) in prokaryotes and eukaryotes. However, annotation of novel functional uORFs is challenging due to their short size of usually <100 codons. While transcription- and translation-level next-generation sequencing methods can be used for genome-wide functional uORF identification, this data are not available for the vast majority of species with sequenced genomes. At the same time, the exponentially increasing amount of genome assemblies gives us the opportunity to take advantage of evolutionary conservation in our predictions of functional ORFs.Here, we present a tool for conserved uORF annotation in 5' upstream sequences of a user-defined protein of interest or a set of protein homologs. It can also be used to find small conserved ORFs within a set of nucleotide sequences. The output includes publication-quality figures with multiple sequence alignments, sequence logos, and locus annotation of the predicted conserved uORFs in graphical vector format., Availability and Implementation: uORF4u is written in Python3 and runs on Linux and MacOS. The command-line interface covers most practical use cases, while the provided Python API allows usage within a Python program and additional customization. Source code is available from the GitHub page: github.com/GCA-VH-lab/uorf4u. Detailed documentation that includes an example-driven guide available at the software home page: gca-vh-lab.github.io/uorf4u. A web version of uORF4u is available at server.atkinson-lab.com/uorf4u., (© The Author(s) 2023. Published by Oxford University Press.)

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of 'long'-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis., (© 2022. The Author(s).)

Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB 2 :Rel NTD 2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.

Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages 1-3 . Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns 4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRel SJ46 , a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRel SJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRel SJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict' 5 , revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts 6-10 , our results reveal a deeply conserved facet of immunity., (© 2022. The Author(s).)

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site., (© 2022. The Author(s).)

The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that-by analogy to other proteins of this type-may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaA LC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.

Summary: Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (standing for Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web tool version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run., Availability and Implementation: FlaGs can be downloaded from https://github.com/GCA-VH-lab/FlaGs or run online at http://www.webflags.se/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system that uses a holin membrane protein in tandem with a cell wall-editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall-editing enzymes involved was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS)., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antibiotic resistance is mediated through several distinct mechanisms, most of which are relatively well understood and the clinical importance of which has long been recognized. Until very recently, neither of these statements was readily applicable to the class of resistance mechanism known as target protection, a phenomenon whereby a resistance protein physically associates with an antibiotic target to rescue it from antibiotic-mediated inhibition. In this Review, we summarize recent progress in understanding the nature and importance of target protection. In particular, we describe the molecular basis of the known target protection systems, emphasizing that target protection does not involve a single, uniform mechanism but is instead brought about in several mechanistically distinct ways.

The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co-occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) - a σ 54 -transcriptional activator - is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ 54 -promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome., (Copyright © 2020 Takada, Roghanian, Murina, Dzhygyr, Murayama, Akanuma, Atkinson, Garcia-Pino and Hauryliuk.)

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon- or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis , the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (S A ) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ 2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery)., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as an RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA:RelQ complex. RelQ's allosteric regulator, pppGpp, destabilizes RNA binding and activates RelQ's enzymatic activity. Since SAS enzymes are widely distributed in bacteria, and, as has been discovered recently, are also mobilized by phages (Dedrick et al. 2017), RNA binding to SASs could be a widespread mechanism. The initial discovery raises numerous questions regarding RNA-binding function of the SAS enzymes: What is the molecular mechanism underlying the incompatibility of RNA:SAS complex formation with pppGpp binding and (p)ppGpp synthesis? What are the RNA targets in living cells? What is the regulatory output of the system - (p)ppGpp synthesis, modulation of RNA structure and function, or both?

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.

The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p)ppGpp metabolism and (p)ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp. We also discuss the structural basis of transcriptional regulation by (p)ppGpp and the role of (p)ppGpp in GTP metabolism and in the emergence of bacterial persisters.

Background: The ribosome translates mRNA to protein with the aid of a number of accessory protein factors. Translational GTPases (trGTPases) are an integral part of the 'core set' of essential translational factors, and are some of the most conserved proteins across life. This study takes advantage of the wealth of available genomic data, along with novel functional information that has come to light for a number of trGTPases to address the full evolutionary and functional diversity of this superfamily across all domains of life., Results: Through sensitive sequence searching combined with phylogenetic analysis, 57 distinct subfamilies of trGTPases are identified: 14 bacterial, 7 archaeal and 35 eukaryotic (of which 21 are known or predicted to be organellar). The results uncover the functional evolution of trGTPases from before the last common ancestor of life on earth to the current day., Conclusions: While some trGTPases are universal, others are limited to certain taxa, suggesting lineage-specific translational control mechanisms that exist on a base of core factors. These lineage-specific features may give organisms the ability to tune their translation machinery to respond to their environment. Only a fraction of the diversity of the trGTPase superfamily has been subjected to experimental analyses; this comprehensive classification brings to light novel and overlooked translation factors that are worthy of further investigation.

Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNA(Val) with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNA(Val) with valine and preventing formation of mischarged Thr-tRNA(Val) as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.

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.

The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes., (Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The GTPase eEF1A is the eukaryotic factor responsible for the essential, universal function of aminoacyl-tRNA delivery to the ribosome. Surprisingly, eEF1A is not universally present in eukaryotes, being replaced by the paralog EFL independently in multiple lineages. The driving force behind this unusually frequent replacement is poorly understood., Results: Through sequence searching of genomic and EST databases, we find a striking association of eEF1A replacement by EFL and loss of eEF1A's guanine exchange factor, eEF1Bα, suggesting that EFL is able to spontaneously recharge with GTP. Sequence conservation and homology modeling analyses indicate several sequence regions that may be responsible for EFL's lack of requirement for eEF1Bα., Conclusions: We propose that the unusual pattern of eEF1A, eEF1Bα and EFL presence and absence can be explained by a ratchet-like process: if either eEF1A or eEF1Bα diverges beyond functionality in the presence of EFL, the system is unable to return to the ancestral, eEF1A:eEFBα-driven state.

The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.

Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.