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Aminoacyl tRNA synthetases are enzymes involved in the key process of coupling an amino acid to its cognate tRNA. AN3365 is a novel antibiotic that specifically targets leucyl-tRNA synthetase, whose development was halted after evaluation in phase II clinical trials owing to the rapid selection of resistance. In an attempt to bring AN3365 back into the developmental pipeline we have evaluated the

Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p)ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for re

The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecula

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 insight

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycl

The transition of Escherichia coli from the exponential into the stationary phase of growth induces the stringent response, which is mediated by the rapid accumulation of the alarmone nucleotide (p)ppGpp produced by the enzyme RelA. The significance of RelA's functionality during the transition in the opposite direction, i.e. from the stationary phase into new exponential growth, is less well unde

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 pro

Persisters—a drug-tolerant sub-population in an isogenic bacterial culture—have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term “persistence” is currently

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 w

The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf)

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 o

Mitochondrial translation is essentially bacteria-like, reflecting the bacterial endosymbiotic ancestry of the eukaryotic organelle. However, unlike the translation system of its bacterial ancestors, mitochondrial translation is limited to just a few mRNAs, mainly coding for components of the respiratory complex. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in ba

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in inta

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

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to cha

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture

No-go decay (NGD) targets mRNAs with stalls in translation elongation for endonucleolytic cleavage in a process involving the Dom34 and Hbs1 proteins. The crystal structure of a Schizosaccharomyces pombe Dom34-Hbs1 complex reveals an overall shape similar to that of eRF1-eRF3-GTP and EF-Tu-tRNA-GDPNP. Similarly to eRF1 and GTP binding to eRF3, Dom34 and GTP bind to Hbs1 with strong cooperativity,

Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6-Å resolution. This atomic model allow

Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that