Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation

Basu, Ritwika; Sherman, Michael B; Gagnon, Matthieu G.

doi:10.1038/s41467-022-31129-2

Cited by 15 publications

(10 citation statements)

References 56 publications

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“…6b ). The anticodon bases of the fMet-tRNA fMet are perfectly Watson-Crick base paired with the AUG codon of the mRNA in the P-site, representing an elongation competent 70S IC, ready to progress to the elongation cycle 32 . In fact, the observed distance between bound THB and fMet-tRNA fMet is more than 20 Å, which explains why THB does not have any influence on P-site binding of the initiator tRNA or its reactivity to puromycin, supporting our biochemical results that THB does not inhibit initiation (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Parajuli

Emmerich²,

Mandava³

et al. 2023

Nat Commun

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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.

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Section: Resultsmentioning

confidence: 99%

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

Parajuli

Emmerich²,

Mandava³

et al. 2023

Nat Commun

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“…Since H1, H94, and H98 play a role in the stability of the E. coli LSU, we wondered whether these helices play similar roles in other bacterial ribosomes. We examined the region surrounding H1 in various high resolution structures of bacterial ribosomes that contain H98 (Table 1) (32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Out of the surveyed structures, the ribosomes from two organisms, P. aeruginosa and E. faecalis, have interactions between H1, H94, and H98 that form the same structure as in the E. coli ribosome.…”

Section: Discussionmentioning

confidence: 99%

Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Nissley

Kamal

Cate

2023

Preprint

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The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding.In vivoassembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and functionin vitro. RNA tags in theEscherichia colilarge ribosomal (50S) subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth orin vitroribosome activity. Here, we find thatE. coli50S subunits with MS2 tags inserted in H98 are destabilized compared to wild type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

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“…The E. coli 70S ribosomes, isolated from strain MRE600, were prepared as previously described with some modifications (32). Briefly, the cells were washed in buffer containing 20 mM Tris-HCl pH 7.4, 10.5 mM MgCl 2 , 100 mM NH 4 Cl, 0.5 mM EDTA, 6 mM β-mercaptoethanol, and DNase I (16 U/g cells) and then lysed using a microfluidizer LM20-30 (Microfluidics, Westwood, MA).…”

Section: Methodsmentioning

confidence: 99%

Section: Sample Preparation For Cryo-em Data Acquisition and Image Pr...mentioning

confidence: 99%

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin

Paranjpe

Marina

Grachev

et al. 2022

Preprint

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Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

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Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation

Cited by 15 publications

References 56 publications

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

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

Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin

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