Mechanistic insights into the alternative ribosome recycling by HflXr

Seely, Savannah M; Basu, Ritwika S; Gagnon, Matthieu G

doi:10.1093/nar/gkae128

Cited by 5 publications

(3 citation statements)

References 87 publications

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“…The cryo-EM structure of its assembly intermediate with the human mitochondrial 39S large ribosomal subunit resembles the 50S-HflX-A state, with its HD reaching the PTC in an extended state, while its NTE is disordered. Recent time-resolved cryo-EM (TRCEM) studies on Eco and Lmo capture the initial, intermediate, and final states during 70S splitting by HflX, but do not report disordering of the rRNA helices in the resulting LSU 10, 11 (supplementary text, fig. S12).…”

Section: Resultsmentioning

confidence: 99%

“…The ribosomal Sb, known to be a dynamic structure that interacts with multiple translational factors [16][17][18][19][20][21][22] , engages with the GD (Figure 1g). The GD-Switch I interacts with H89 (Figure 1g), potentially to recognize the GTP-bound state of the protein-an essential step for ribosome binding of HflX 1,3,5,10,11 . SSU head rotation along with tRNA transition to P/E state facilitate HflX-ND1 binding, with concomitant interaction of HflX-CTD with the Sb (Figure 1e-g).…”

Section: Structures Of the Mycobacterial 70s-hflx And 50s-hflxmentioning

confidence: 99%

“…Mycobacterial HflX possesses a species-specific NTE (39 amino acid in Msm) and a variable-length insertion (9 amino acids in Msm) in the HflX-HD 1 . Timeresolved cryogenic electron microscopy (TRCEM) of HflX-mediated ribosome splitting in Eco 10 and Lmo 11 suggests that rotation of SSU enabled by HflX leads to inter-subunit bridge disruption and 70S splitting. Mycobacterial ribosomes have two additional intersubunit bridges [12][13][14][15] formed by SSU proteins bS6 and bS22 interaction with H54a and H70, respectively, which may influence their splitting mechanism.…”

Section: Introductionmentioning

confidence: 99%

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Drug resistance through ribosome splitting and rRNA disordering in mycobacteria

Majumdar,

Kashyap,

Koripella

et al. 2024

Preprint

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HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX- mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic- bound inactive 50S subunits.

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Section: Resultsmentioning

confidence: 99%

Section: Structures Of the Mycobacterial 70s-hflx And 50s-hflxmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria

Majumdar,

Kashyap,

Koripella

et al. 2024

Preprint

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Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria

Srinivasan,

Banerjee,

Sengupta

2024

Structure

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RqcH supports survival in the absence of non-stop ribosome rescue factors

Callan,

Prince,

Feaga

2024

Preprint

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Ribosomes frequently translate truncated or damaged mRNAs due to the extremely short half-life of mRNAs in bacteria. When ribosomes translate mRNA that lacks a stop codon (non-stop mRNA), specialized pathways are required to rescue the ribosome from the 3’ end of the mRNA. The most highly conserved non-stop rescue pathway istrans-translation, which is found in greater than 95% of bacterial genomes. In all Proteobacteria that have been studied, the alternative non-stop ribosome rescue factors, ArfA and ArfB, are essential in the absence oftrans-translation. Here, we investigate the interaction between non-stop rescue pathways and RqcH, a ribosome quality control factor that is broadly conserved outside of Proteobacteria. RqcH does not act directly on non-stop ribosomes but adds a degron tag to stalled peptides that obstruct the large ribosomal subunit, which allows the stalled peptide to be cleared from the ribosome by peptidyl-tRNA hydrolase (PTH). We show thatBacillus subtiliscan survive withouttrans-translation and BrfA (Bacillus ArfA homolog), due to the presence of RqcH. We also show that expression of RqcH and its helper protein RqcP rescues the synthetic lethality of ΔssrAΔarfAinEscherichia coli. These results suggest that non-stop ribosome complexes can be disassembled and then cleared because of the tagging activity of RqcH, and that this process is essential in the absence of non-stop ribosome rescue pathways. Moreover, we surveyed the conservation of ribosome rescue pathways in >14,000 bacterial genomes. Our analysis reveals a broad distribution of non-stop rescue pathways, especiallytrans-translation and RqcH, and a strong co-occurrence between the ribosome splitting factor MutS2 and RqcH. Altogether, our results support a role for RqcH in non-stop ribosome rescue and provide a broad survey of ribosome rescue pathways in diverse bacterial species.ImportanceRibosome stalling on damaged mRNA is a major problem in bacteria. It is estimated that 2-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes, such astrans-translation, are often essential for viability. We investigated the functional overlap between RqcH and the non-stop ribosome rescue systems (ArfA andtrans-translation) that are present in bothE. coliandB. subtilis. Since these two species are extremely distant relatives, our work is likely to have wider implications for understanding ribosome rescue in bacteria. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >14,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.

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Mechanistic insights into the alternative ribosome recycling by HflXr

Cited by 5 publications

References 87 publications

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria

Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria

RqcH supports survival in the absence of non-stop ribosome rescue factors

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