Assembly and functionality of the ribosome with tethered subunits

Aleksashin, Nikolay A.; Leppik, Margus; Hockenberry, Adam J.; Klepacki, Dorota; Vázquez‐Laslop, Nora; Jewett, Michael C.; Rèmme, Jaanus; Mankin, Alexander S.

doi:10.1038/s41467-019-08892-w

Cited by 42 publications

(44 citation statements)

References 61 publications

(86 reference statements)

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“…These results are consistent with a previous ribosome profiling analysis in Ribo-Texpressing cells, which highlighted an increase in ribosome density at start codons and close to the 3' ends of genes 3 . The tethering of the ribosomal subunits had mild effects on the initiation and termination/recycling steps of translation.…”

Section: Discussionsupporting

confidence: 93%

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Translational accuracy of a tethered ribosome

Fabret

Namy

2020

Preprint

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Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli 1 . A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior in -1 or +1 frameshifting, stop codon readthrough, and internal translation initiation.Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.

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Section: Discussionsupporting

confidence: 93%

“…A ribosome with tethered subunits, in which the two subunits were covalently linked in a single entity, was recently engineered in Escherichia coli 1,3 . The hybrid ribosomal RNA (rRNA) consisted of the small and large subunit rRNA sequences, linked by short RNA linkers.…”

Section: Introductionmentioning

confidence: 99%

Translational accuracy of a tethered ribosome

Fabret

Namy

2020

Preprint

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“…However, while achieving full orthogonality, the unusual design of Ribo-T limits its functionality. Ribo-T translates proteins with only half the rate of the dissociable ribosome 14 and it is more slow in departing from the start codons in comparison with the wt ribosomes 18 . Furthermore, the biogenesis of even non-orthogonal Ribo-T is rather slow and inefficient 18 and the assembly problems could be further exacerbated if the ribosome's functional centers are subjected to additional alterations 8 .…”

Section: Ribo-tmentioning

confidence: 99%

A fully orthogonal system for protein synthesis in bacterial cells

et al. 2020

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Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

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“…Indeed, some of these engineered orthogonal ribosomes are similarly affected by assembly defects. 11 Although P7A7…”

Section: Discussionmentioning

confidence: 99%

“…New sequence-defined polymerization chemistries will likely require repurposing a suite of enzymes and nucleic acids, including tRNAs, 2,3 aminoacyl tRNA synthetases (aaRSs), 4,5 elongation factor Tu (EF-Tu), 6,7 in addition to the ribosome itself. [8][9][10][11][12] The catalytic core of the ribosome, the peptidyl transferase center (PTC), induces the attack of an A-site aminoacyl-tRNA on the ester bond of the P-site peptidyl-tRNA, transferring and extending the growing polypeptide chain. The PTC is thought to encourage proper polymerization through several mechanisms including orienting the substrates, 13 favoring productive acid-base chemistry, 14 and protecting the peptidyl-tRNA from dead-end hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Defects in the assembly of ribosomes selected for β-amino acid incorporation

Ward

Watson

et al. 2019

Preprint

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Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy.Although P7A7 is a functional ribosome in vivo , it is inactive in vitro , and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

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Assembly and functionality of the ribosome with tethered subunits

Cited by 42 publications

References 61 publications

Translational accuracy of a tethered ribosome

Translational accuracy of a tethered ribosome

A fully orthogonal system for protein synthesis in bacterial cells

Defects in the assembly of ribosomes selected for β-amino acid incorporation

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