Biology subjects

Metelev, M.

Publications and source records attributed to Metelev, M..

6 recordsLinked to original sources

bioRxiv2026

16S rRNA Modifications Are Dispensable for Viability but Collectively Optimize Ribosome Biogenesis and Translation Initiation in Escherichia coli

Ribosomal RNAs contain numerous conserved nucleotide modifications, yet the functional importance of most of these modifications remains unclear. In Escherichia coli, deletion of individual 16S rRNA modification enzymes generally produces only minor phenotypes, raising questions about their biological significance. Here, we generated a comprehensive collection of deletion mutants lacking individual and combined 16S rRNA modifications, culminating in a strain lacking all known 30S ribosomal subunit modifications. Despite the absence of all known 16S rRNA modifications, cells remained viable, exhibiting a fitness defect of ~30% at 37 {degrees}C that increased to ~50% at 20 {degrees}C, consistent with impaired ribosome biogenesis. We identified strong epistatic interactions between modifications in the 3' major and 3' minor domains of 16S rRNA, resulting in disproportionately large effects on both fitness and antibiotic susceptibility. Live-cell single-molecule tracking revealed a marked increase in the fraction of non-translating ribosomes and a prolonged time required to enter productive translation, whereas translational elongation by actively engaged 70S ribosomes remained largely unaffected. In addition, fluorescence-based measurements showed that unmodified ribosomes exhibited increased stringency during translation initiation, reducing utilization of near-cognate start codons. Together, these findings demonstrate that 16S rRNA modifications are not essential for viability but collectively enhance the efficiency, robustness, and fidelity of ribosome assembly and translational initiation.

microbiology↗

bioRxiv2026

Ribosome collisions trigger tmRNA-mediated rescue through mRNA disengagement

Ribosome rescue pathways maintain translation during normal growth and contribute to stress tolerance. In Escherichia coli, HrpA is a ribosome-collision recognition factor that mediates antibiotic tolerance by resolving stalled ribosomes, yet the fate of the resulting ribosomal intermediate, with a topologically trapped peptidyl-tRNA in the 50S nascent peptide exit tunnel, has remained unclear. Using in vivo single-molecule tracking, we show that under antibiotic treatment, HrpA-dependent collision processing promotes recruitment of tmRNA and Hsp15 as downstream rescue factors. Cryo-electron microscopy of affinity-purified native complexes from tmRNA-deficient cells reveals accumulation of aberrant 70S{middle dot}peptidyl-tRNA complexes and shows that Hsp15, previously thought to interact exclusively with 50S{middle dot}peptidyl-tRNA, binds to 70S complexes lacking mRNA in the E site. This intermediate can be resolved by the canonical tmRNA pathway, thereby connecting HrpA-dependent collision processing to canonical tmRNA-mediated rescue.

molecular biology↗

bioRxiv2024

A complex between IF2 and NusA links transcription and translation

The main function of translation initiation factors is to assist ribosomes in selecting the correct reading frame on an mRNA. This process has been extensively studied with the help of reconstituted in vitro systems, but the dynamics in living cells have not been characterized. In this study, we performed single-molecule tracking of the bacterial initiation factors IF2, IF3, as well as the initiator fMet-tRNAfMet directly in growing Escherichia coli cells. Our results reveal the kinetics of factor association with the ribosome and, among other things, highlight the respective antagonistic roles of IF2 and IF3 in the process. Importantly, our comparisons of in vivo binding kinetics of the two naturally occurring isoforms of IF2 reveal that the longer IF2 isoform directly interacts with the transcriptional factor NusA, a finding further corroborated by pull-down and cross-linking experiments. We further show that cells with compromised binding between IF2 and NusA display slow adaptation to new growth conditions. Taken together, these results suggest a novel coupling mechanism between transcription and translation initiation, important for the rewiring of the transcriptome during a shift of growth media.

molecular biology↗

bioRxiv2024

Dynamic binding of the bacterial chaperone Trigger factor to translating ribosomes

The bacterial chaperone Trigger factor (TF) binds to ribosome-nascent chain complexes (RNCs) and co-translationally aids the folding of proteins in bacteria. Decades of studies have given a broad, but often conflicting, description of the substrate specificity of TF, its RNC-binding dynamics, and competition with other RNC-binding factors, such as the Signal Recognition Particle (SRP). Previous RNC-binding kinetics experiments were conducted on stalled RNCs in reconstituted systems, and consequently, may not represent the interaction of TF with ribosomes translating mRNA in the cytoplasm of the cell. Here, we used single-particle tracking (SPT) to measure TF binding to actively translating ribosomes inside living Escherichia coli. In cells, TF displays two distinct binding modes -- long (ca 1 s) target-specific RNC binding, and shorter (ca 50 ms) sampling of non-target RNCs. RNC binding events are interrupted only by transient excursions to a freely diffusing state (ca 40 ms). We also show that TF competes with SRP for RNC binding in vivo, and in doing so, tunes the binding selectivity of SRP.

biophysics↗

bioRxiv2020

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.

molecular biology↗

bioRxiv2019

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

molecular biology↗