Biology subjects

Selmer, M.

Publications and source records attributed to Selmer, M..

10 recordsLinked to original sources

bioRxiv2026

Structural characterization of ribosome recycling and fusidic acid inhibition in Staphylococcus aureus

During bacterial ribosome recycling, 70S ribosomes are split into subunits by ribosome recycling factor (RRF) and elongation factor G (EF-G). The antibiotic fusidic acid (FA) inhibits elongation and ribosome recycling by locking EF-G to the ribosome. Yet, no functional ribosome recycling FA complex has been successfully captured. Here we used single-particle cryo-electron microscopy to resolve multiple FA-stalled intermediates of Staphylococcus aureus ribosomes, including a previously unobserved complex containing both RRF and EF-G. Our structures reveal how RRF and EF-G jointly disrupt inter-subunit bridges, promote back-rotation of the small subunit, and destabilize the tRNA to facilitate ribosome splitting. We further show that FA predominantly inhibits recycling by trapping EF-G on the post-termination complex in the absence of RRF, preventing formation of the active RRF*EF-G complex. These insights advance understanding of the molecular mechanism of bacterial ribosome recycling and the mode of action of FA as an antibiotic. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/721003v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@12951c2org.highwire.dtl.DTLVardef@1b6b7f3org.highwire.dtl.DTLVardef@e6cc04org.highwire.dtl.DTLVardef@5c4521_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

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↗

bioRxiv2026

23S rRNA modifications stimulate catalytic activity and prevent the formation of alternative structures

Ribosomal RNA modifications cluster around the peptidyl transferase center (PTC), the catalytic center of the ribosome, yet their collective functional roles remain unclear. Here we analyze Escherichia coli ribosomes lacking 11 or 12 modifications near the PTC. Using kinetic assays, we show these hypo-modified ribosomes catalyze peptide bond formation at rates two- to threefold lower than wild-type and exhibit reduced thermal stability. Cryo-electron microscopy of hypo-modified ribosomes reveals multiple alternative conformations of the PTC and exit tunnel regions, disrupting native stacking and hydrogen bonding critical for positioning of tRNA substrates. These findings indicate that rRNA modifications stabilize the native PTC structure, preventing formation of alternative, nonfunctional conformations and thereby enhance catalytic efficiency. Our study provides insight into how rRNA modifications fine-tune ribosome function by maintaining structural integrity essential for efficient translation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/708810v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f06f16org.highwire.dtl.DTLVardef@d189aorg.highwire.dtl.DTLVardef@1ca55acorg.highwire.dtl.DTLVardef@bec055_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Doublet decoding of tRNASer3 demonstrates plasticity of ribosomal decoding center

Frameshifts can be caused by specific combinations of tRNA and mRNA. The wildtype AGC-decoding E. coli tRNASer3GCU was in the 1980s shown to induce -1 ribosomal frameshifting on GCA alanine codons, and proposed to read a two-base codon instead of a canonical triplet. It has remained unclear whether this type of non-cognate decoding can be accommodated by the ribosome. We here performed single-particle cryo-EM reconstructions on E. coli 70S ribosomes with the frameshift-inducing tRNASer3 bound to the non-cognate GCA codon or the cognate AGC codon in the ribosomal A site. The structures demonstrate that doublet-decoding is made possible when A1493, the conserved monitoring base in 16S rRNA, mimics a first codon base, forming a Hoogsteen base pair with U36 from the anticodon and stacking with the mRNA. This interaction pushes the first two bases of the A-site codon in position for base pairing with C35 and G34 of the anticodon.

molecular biology↗

bioRxiv2024

Structural mechanism of FusB-mediated rescue from fusidic acid inhibition of protein synthesis

Antibiotic resistance protein FusB rescues protein synthesis from inhibition by fusidic acid (FA), which locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis. Here, we present time-resolved single-particle cryo-EM structures explaining the mechanism of FusB-mediated rescue. FusB binds to the FA-trapped EF-G on the ribosome, causing large-scale conformational changes of EF-G that break ribosome interactions. This leads to dissociation of EF-G from the ribosome, followed by FA release. We also observe two independent binding sites of FusB on the classical-state ribosome, overlapping with the binding site of EF-G to each of the ribosomal subunits, yet not inhibiting tRNA delivery. Our results reveal an intricate resistance mechanism involving specific interactions of FusB with both EF-G and the ribosome, and a non-canonical release pathway of EF-G.

molecular biology↗

bioRxiv2023

Structures of the Staphylococcus aureus ribosome inhibited by fusidic acid and fusidic acid cyclopentane

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 [A] resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 [A] resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric hybrid state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.

molecular biology↗

bioRxiv2023

Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis

Bacteriophage T3 encodes a SAMase that through cleavage of S-adenosyl-methionine (SAM) circumvents the SAM-dependent Type I Restriction-Modification defence of the host bacterium Escherichia coli. Here, we show that the SAMase also allows T3 to evade BREX defence. SAM degradation weakly affects BREX methylation of host DNA, but completely inhibits the defensive function of BREX, suggesting that SAM is required as a co-factor for BREX-mediated exclusion of phage DNA. The anti-BREX activity of the T3 SAMase is mediated by two independent mechanisms: enzymatic degradation of SAM and downregulation of SAM synthesis through direct inhibition of the host SAM synthase MetK. We determined a 2.8 [A] cryo-EM structure of the eight-subunit T3 SAMase-MetK complex. Structure guided mutagenesis of the SAMase-MetK interface revealed that the interaction with MetK stabilizes the T3 SAMase in vivo, thus further stimulating its anti-BREX activity. This work provides insights in the versatility and intricacy of bacteriophage counter-defence mechanisms and highlights the role of SAM as an important co-factor of diverse phage-defence systems.

microbiology↗

bioRxiv2022

Cryo-EM reconstructions of LiCl core particles show strong similarities between disassembly and assembly of the ribosomal 50S subunit

Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were early shown competent for in vitro assembly into functional 50S subunits. We here used cryo-EM to determine the structure of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variation in extent of ordered 23S rRNA and occupancy of r-proteins could be identified, and resolved to between 2.8 [A] and 9 [A] resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed ordered density for 23S rRNA domains 0, I, II, III, VI and the 5-half of domain IV. In addition, smaller core particles were discovered, which show that the most stable part of the 50S under high-salt conditions includes parts of domain 0 and most of domains I, III and the 5-half of domain IV and four to eight r-proteins. Our data support a multi-pathway disassembly process based on independent folding blocks, similar but reverse to the assembly process. The study provides examples of dependencies between complex tertiary RNA structure and RNA-protein interactions where protein extensions dissociate before the globular domains. We observe formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structure and prevent non-native interactions also after folding. IMPORTANCERibosome assembly and stability remain only partially understood. Incubation of ribosomes with salts was early shown to induce dissociation of the more loosely bound ribosomal proteins (r-proteins) and formation of so-called core particles. In this work, cryo-EM imaging of 50S LiCl core particles from E. coli for the first time allowed structural characterization of such particles of different size. The smallest particles demonstrate what constitutes the smallest stable core of the 50S ribosomal subunit, and the sequential comparison with larger particles show how the ribosome disassembles and assembles in layers of rRNA structure stabilized by globular domains and extended tails of r-proteins. Major insights are that ribosomes disassemble along different paths, that dissociation of r-proteins can induce misfolding of rRNA and that extended tails of r-proteins dissociate from rRNA before the globular domains. The characterized particles can be used in future mechanistic studies of ribosome biogenesis.

molecular biology↗

bioRxiv2020

Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family

The first SAM degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as an S-adenosyl-L-methionine (SAM) hydrolase forming 5-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha-beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography and NMR spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis, in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

biochemistry↗

bioRxiv2020

Structural recognition of spectinomycin by resistance enzyme ANT(9) from Enterococcus faecalis

Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is the modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT(9) family or by the dual-specificity ANT(3")(9) family that also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3")(9) AadA from Salmonella enterica. ANT(9) from Enterococcus faecalis adenylates the 9-hydroxyl of spectinomycin. We here present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9) in apo form, in complex with ATP, spectinomycin and magnesium or in complex with only spectinomycin. ANT(9) shows similar overall structure as AadA with an N-terminal nucleotidyltransferase domain and a C-terminal -helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates by 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight 5 helix and a shorter 5-6 loop that would clash with the larger streptomycin substrate. In the active site, we observe two magnesium ions, one of them in a previously un-observed position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that also spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, will be subjected to modification by ANT(9) and ANT(3")(9) enzymes.

biochemistry↗