ArfB can displace mRNA to rescue stalled ribosomes

Carbone, C.E.; Demo, Gabriel; Madireddy, Rohini; Svidritskiy, Egor; Коростелев, А.A.

doi:10.1038/s41467-020-19370-z

Cited by 18 publications

(40 citation statements)

References 72 publications

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“…A recent cryo-EM study revealed two intermediate steps of ArfB accommodation to a non-stop complex ( Carbone et al, 2020 ). In the first intermediate the C-terminal tail has already bound to the mRNA entry channel and formed an α-helix, while the NTD is associated with H69 of the 23S rRNA and the flexible GGQ loop is pointed away from the A-site of the 50S subunit ( Figures 4A,B ).…”

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

“…The second intermediate shows that the NTD is still bound to H69, but is rotated so that the GGQ loop is directed toward the large subunit A-site ( Figure 4C ), priming ArfB for insertion into the PTC to perform hydrolysis. Eventually, the NTD is placed in the A-site of the large subunit and accommodates into the PTC ( Figure 4D ), which is induced upon binding of the NTD ( Gagnon et al, 2012 ; Carbone et al, 2020 ; Chan et al, 2020 ). The GGQ motif adopts an analogous conformation to the GGQ motif of bacterial class I RFs and mediates hydrolysis of the ester bond between the peptide and the P-tRNA.…”

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

“…(A) Non-stop complex (large subunit, 50S, gray; small subunit, 30S, yellow) with peptidyl-tRNA (green) in the P-site and mRNA extending two nucleotides into the A-site (short mRNA, cyan) [PDB ID 7JSZ ( Carbone et al, 2020 )]. (B-D) ArfB (purple) bound to the non-stop complex with the NTD in the collapsed (B) [PDB ID 7JSZ ( Carbone et al, 2020 )], pre-accommodated (C) [PDB ID 7JSW ( Carbone et al, 2020 )], as well as fully accommodated (D) [PDB ID 6YSS ( Chan et al, 2020 )] conformation. The conformation of the NTD is enlarged in the inlays.…”

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

“…(E) Ribosome with mRNA extending nine nucleotides into the A-site and the mRNA channel (long mRNA, cyan) and peptidyl-tRNA in the P-site [PDB ID 6YSR ( Chan et al, 2020 )]. (F) ArfB dimer (ArfB, purple and ArfB-2, pale violet red) on the ribosome with the long mRNA sandwiched between the ArfB dimer and the P-site tRNA [PDB ID 7JT1 ( Carbone et al, 2020 )]. (G) ArfB or the ArfB dimer dissociated and ribosome recycling had occurred.…”

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

“…during termination ( Korostelev et al, 2008 ; Laurberg et al, 2008 ; Weixlbaumer et al, 2008 ; Korostelev et al, 2010 ), and in the presence of A-site tRNA ( Arenz et al, 2016 ), ArfA/RF2 ( James et al, 2016 ; Demo et al, 2017 ; Huter et al, 2017c ; Ma et al, 2017 ; Zeng et al, 2017 ), or SmpB⋅tmRNA ( Rae et al, 2019 )], respectively flips out of H69 ( Gagnon et al, 2012 ; Chan et al, 2020 ). Interestingly, Korostelev and co-workers formed a non-stop complex with two mRNA nucleotides reaching into the A-site ( Carbone et al, 2020 ). In this complex the decoding center adopts a different conformation in which A1492 and A1493 are flipped into h44 and stack with the overhanging mRNA nucleotides.…”

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

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Ribosome Rescue Pathways in Bacteria

2021

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Ribosomes that become stalled on truncated or damaged mRNAs during protein synthesis must be rescued for the cell to survive. Bacteria have evolved a diverse array of rescue pathways to remove the stalled ribosomes from the aberrant mRNA and return them to the free pool of actively translating ribosomes. In addition, some of these pathways target the damaged mRNA and the incomplete nascent polypeptide chain for degradation. This review highlights the recent developments in our mechanistic understanding of bacterial ribosomal rescue systems, including drop-off, trans-translation mediated by transfer-messenger RNA and small protein B, ribosome rescue by the alternative rescue factors ArfA and ArfB, as well as Bacillus ribosome rescue factor A, an additional rescue system found in some Gram-positive bacteria, such as Bacillus subtilis. Finally, we discuss the recent findings of ribosome-associated quality control in particular bacterial lineages mediated by RqcH and RqcP. The importance of rescue pathways for bacterial survival suggests they may represent novel targets for the development of new antimicrobial agents against multi-drug resistant pathogenic bacteria.

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Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

Section: Bacterial Ribosome Rescue Systemsmentioning

confidence: 99%

See 3 more Smart Citations

Ribosome Rescue Pathways in Bacteria

2021

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Efficient In Vitro Full‐Sense‐Codons Protein Synthesis

Tang

Wang

et al. 2022

Advanced Biology

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orthogonal suppressor tRNA holds the key to the incorporation efficiency. Recently, improved incorporation is achieved in E. coli strain C321.ΔA, in which all amber codons, accounting for 8% of stop codons in E. coli, [1] are replaced and prfA is removed away from the genome. [2] However, it will be extremely lethal for a cell to simultaneously interfere with all three stop codons. Saturation mutagenesis for molecular evolution is another application restricted by termination mechanisms. Stop codon involved in NNN randomized codon causes undesired premature termination. One stop codon is still generated even with NNK or NNS, moreover, the reduced codon set excludes codons with high usage frequency (Figure S1, Supporting Information). We anticipate that complete codon-dependent termination defect protein translation could efficiently liberate all codons for sense function and improve all the genetic code engineering applications (Figure 1).In the cell, termination of translation includes both the essential mechanism of class-I release factors and alternative mechanisms, [3] such as tmRNA (ssrA) mediated transtranslation [4] and translation stalling rescue. [5] As alternative termination ArfT in F. tularensis, [6] ResQ in Bacillus subtilis [7] are reported. In contrast to RF1, there is still no feasible approach for deletion of RF2, major class-I release factor in charge of the essential termination and plays a critical role in alternative terminations as well, i.e., post-peptidyl transfer quality control and alternative ribosome rescue. [8] However, cell viability becomes a barrier for in vivo assessment of global termination function. Therefore, there is strong curiosity about what if losing all essential termination machineries.Essential genes cannot be genetically deleted in a living cell. Pdt peptide containing 27 amino acids in length evolved from Mesoplasma florum Lon protease (mf-Lon) is demonstrated as being able to specifically target protein to efficient degradation in E. coli. [9] Pdt-tag is fused to nascent chain release factor RF1 and RRF respectively in E. coli MG1655pro, but failed on prfB encoding release factor RF2. Thus far, complete removal of all termination functions in either living cell or cell lysate was not yet achieved.Here, we present one efficient in vitro protein synthesis with 64 sense codons (iPSSC). The mf-Lon directed protein degradation is demonstrated as one efficient approach for the Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs...

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