Three-dimensional structure-guided evolution of a ribosome with tethered subunits

Kim, Do Soon; Watkins, Andrew; Bidstrup, Erik; Lee, Joongoo; Topkar, V.V.; Kofman, Camila; Schwarz, Kevin J.; Liu, Yan; Pintilie, Grigore; Roney, Emily; Das, Rhiju; Jewett, Michael C.

doi:10.1038/s41589-022-01064-w

Cited by 12 publications

(16 citation statements)

References 51 publications

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“…Yet, with technological advances, these cell-free systems are now closer to 100% incorporation efficiency 51,52 . We expect ribosome engineering platforms such as in vitro ribosome synthesis and evolution (RISE) 53,54 , and computational methods for RNA secondary structure prediction (e.g., Eterna, Rosetta stepwise Monte Carlo method) [55][56][57][58] could lead to engineered ribosomes that are capable of forming the pyridazinone bond formation more efficiently.…”

Section: Discussionmentioning

confidence: 99%

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

et al. 2022

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The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome’s ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

et al. 2022

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“…In addition, due to primer bias, randomized libraries constructed using PCR are known to have imbalanced initial populations, skewing assessments of a library's members by overemphasizing the more common ones ( 29 ). PCR-based library construction approaches are also difficult to apply to multiple regions of rRNA that are close in three-dimensional space but not primary sequence space, which is common in the structurally complex PTC ( 30 ). A further challenge is that DNA libraries are typically propagated in cells, where transformation idiosyncrasies limit library size ( 11 , 31–33 ).…”

Section: Introductionmentioning

confidence: 99%

Computationally-guided design and selection of high performing ribosomal active site mutants

Kofman

Watkins

Kim

et al. 2022

Nucleic Acids Research

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Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

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“…The catalytic core (i.e., PTC) of the ribosome has already been shown that it can be mutated on the ribosomal RNA (rRNA) level and that such engineered ribosomes [73,157,158] can enhance the incorporation efficiency of β-, D-amino acids, and dipeptides into a polypeptide chain [159][160][161][162][163][164]. Building upon this advancement, we have also demonstrated the incorporation of cyclic γ-amino acids [75] and fluorescent amino acids [83,165] into a peptide using engineered ribosomes. These results suggest that the ribosome's preference toward L-α-amino acids evolutionarily optimized to support life are not critically conserved and the ribosome can be engineered to have new catalytic activity beyond nature's limit.…”

Section: Engineering the Protein Translation Systemsmentioning

confidence: 83%

Cell-free Biosynthesis of Peptidomimetics

Lee

Willi

Cho

et al. 2023

Biotechnol Bioproc E

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A wide variety of peptidomimetics (peptide analogs) possessing innovative biological functions have been brought forth as therapeutic candidates through cell-free protein synthesis (CFPS) systems. A key feature of these peptidomimetic drugs is the use of non-canonical amino acid building blocks with diverse biochemical properties that expand functional diversity. Here, we summarize recent technologies leveraging CFPS platforms to expand the reach of peptidomimetics drugs. We also offer perspectives on engineering the translational machinery that may open new opportunities for expanding genetically encoded chemistry to transform drug discovery practice beyond traditional boundaries.

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Three-dimensional structure-guided evolution of a ribosome with tethered subunits

Cited by 12 publications

References 51 publications

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

Computationally-guided design and selection of high performing ribosomal active site mutants

Cell-free Biosynthesis of Peptidomimetics

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