Engineered ribosomes with tethered subunits for expanding biological function

Carlson, Erik D.; d’Aquino, Anne E.; Kim, Do Soon; Fulk, Emily M.; Hoang, Kim Le Mai; Szal, Teresa; Mankin, Alexander S.; Jewett, Michael C.

doi:10.1038/s41467-019-11427-y

Cited by 59 publications

(69 citation statements)

References 55 publications

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“…2a, b). In the resulting OSYRIS cells, the Ribo-T rRNA with improved 16S-23S tethers 17 is expressed from the optimized pRibo-Tt plasmid. Another plasmid, poRbs, carries the rRNA genes of the dissociable o-ribosomes, whose 16S rRNA gene has an altered ASD ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…a Organization of rRNA genes and structure of the dissociable 70S ribosome (left) and Ribo-T (right). The small and large subunits of Ribo-T are covalently linked by two RNA tethers connecting circularly-permutated 23S rRNA to the loop of helix 44 in 16S rRNA 14,17 . The sequences of 16S rRNA sequences are shown in olive and those of 23S rRNA in gray.…”

Section: Ribo-tmentioning

confidence: 99%

“…designs [15][16][17] , circularly permutated 23S rRNA is embedded into the 16S rRNA, yielding a ribosome whose subunits are tethered by two RNA linkers ( Fig. 1a).…”

Section: Ribo-tmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Ribo-tmentioning

confidence: 99%

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A fully orthogonal system for protein synthesis in bacterial cells

et al. 2020

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“…Ribo-T ribosomes are present, but only the Ribo-T ribosomes can initiate lacZ translation, because of the mutated SD sequence. Crosstalk between wild-type ribosomes and the orthogonal mRNA has recently been described 8 . We checked the level of crosstalk in our system, and found that it was too low to affect the results.…”

Section: Gaccgtaatcatggtatattgtggtgttaaagttaaacaaaattatttc-3'mentioning

confidence: 99%

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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“…Recently, engineering mutant ribosomes to elucidate key principles underpinning the process of translation, program cellular function, and generate novel sequence-defined polymers has emerged as a major opportunity in chemical and synthetic biology [1][2][3][4][5][6][7] . Although efforts to engineer ribosomal function have achieved some success 8,9 , especially with the recent advance of tethered ribosome systems [10][11][12] , a significant repurposing of the translation apparatus is constrained by challenges inherent to altering translation in living cells. A completely in vitro approach could overcome cell viability constraints to repurpose the ribosome into a re-engineered machine for understanding, harnessing, and expanding the capabilities of the translation apparatus.…”

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confidence: 99%

In vitro ribosome synthesis and evolution through ribosome display

et al. 2020

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Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a~1.7 × 10 7 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of~4 × 10 3 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

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Engineered ribosomes with tethered subunits for expanding biological function

Cited by 59 publications

References 55 publications

A fully orthogonal system for protein synthesis in bacterial cells

A fully orthogonal system for protein synthesis in bacterial cells

Translational accuracy of a tethered ribosome

In vitro ribosome synthesis and evolution through ribosome display

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