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Controlling orthogonal ribosome subunit interactions enables evolution of new function.

Schmied WH, Tnimov Z, Uttamapinant C, Rae CD, Fried SD, Chin JW
Nature 564 444-448 (2018)
Cited: 50 times
EuropePMC logo PMID: 30518861

Abstract

Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit1. Directed evolution of orthogonal ribosomes has provided access to new ribosomal function, and the evolved orthogonal ribosomes have enabled the encoding of multiple non-canonical amino acids into proteins2-4. The original orthogonal ribosomes shared the pool of 23S ribosomal RNAs, contained in the large subunit, with endogenous ribosomes. Selectively directing a new 23S rRNA to an orthogonal mRNA, by controlling the association between the orthogonal 16S rRNAs and 23S rRNAs, would enable the evolution of new function in the large subunit. Previous work covalently linked orthogonal 16S rRNA and a circularly permuted 23S rRNA to create orthogonal ribosomes with low activity5,6; however, the linked subunits in these ribosomes do not associate specifically with each other, and mediate translation by associating with endogenous subunits. Here we discover engineered orthogonal 'stapled' ribosomes (with subunits linked through an optimized RNA staple) with activities comparable to that of the parent orthogonal ribosome; they minimize association with endogenous subunits and mediate translation of orthogonal mRNAs through the association of stapled subunits. We evolve cells with genomically encoded stapled ribosomes as the sole ribosomes, which support cellular growth at similar rates to natural ribosomes. Moreover, we visualize the engineered stapled ribosome structure by cryo-electron microscopy at 3.0 Å, revealing how the staple links the subunits and controls their association. We demonstrate the utility of controlling subunit association by evolving orthogonal stapled ribosomes which efficiently polymerize a sequence of monomers that the natural ribosome is intrinsically unable to translate. Our work provides a foundation for evolving the rRNA of the entire orthogonal ribosome for the encoded cellular synthesis of non-canonical biological polymers7.

Articles - 6hrm mentioned but not cited (2)

  1. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Schmied WH, Tnimov Z, Uttamapinant C, Rae CD, Fried SD, Chin JW. Nature 564 444-448 (2018)
  2. Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity. Tüting C, Iacobucci C, Ihling CH, Kastritis PL, Sinz A. Sci Rep 10 12618 (2020)


Reviews citing this publication (14)

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  29. Airway microecology in rifampicin-resistant and rifampicin-sensitive pulmonary tuberculosis patients. Cai X, Luo Y, Zhang Y, Lin Y, Wu B, Cao Z, Hu Z, Wu X, Tan S. BMC Microbiol 22 286 (2022)
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