Community science designed ribosomes with beneficial phenotypes

Krüger, Antje; Watkins, Andrew; Wellington-Oguri, Roger; Romano, Jonathan; Kofman, Camila; DeFoe, Alysse; Kim, Yejun; Anderson-Lee, Jeff; Fisker, Eli; Townley, Jill; Participants, Eterna; d’Aquino, Anne E.; Das, Rhiju; Jewett, Michael C.

doi:10.1101/2021.09.05.458952

Cited by 5 publications

(5 citation statements)

References 44 publications

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“…We observed an inverse correlation ( r = –0.62, P = 1.3e–06) between performance in iSAT and SWM score (Figure 1B ). Lower scores, which indicate a more stable rRNA structural conformation, were more likely to yield functional ribosomes in iSAT, a result consistent with our recent work ( 51 ). Given this correlation, we moved forward with using SWM score as a metric for selection of successful mutants.…”

Section: Resultssupporting

confidence: 91%

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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Section: Resultssupporting

confidence: 91%

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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show abstract

“…We observed a correlation (r=0.62) between performance in iSAT and SWM score ( Figure 1B ). Lower scores, which indicate a more stable rRNA structural conformation, were more likely to yield active ribosomes, a result consistent with our recent work (44). Given this correlation, we moved forward with using SWM score as a metric for selection of highly active ribosomes.…”

Section: Resultssupporting

confidence: 92%

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

Kofman

Watkins²,

Kim

et al. 2022

Preprint

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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, 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 may serve as a powerful tool for high-resolution rRNA design.

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“…When soluble tdTomato was expressed, we obtained the characteristic peaks of 30S, 50S and 70S, corresponding to the small and large ribosomal subunits and assembled 70S ribosome (magenta trace Fig. 1d )[32], [33]. When MscL was expressed, we observed additional peaks that are assigned to polysomes, i.e., multiple ribosomes stalled along an RNA strand (black trace Fig.…”

Section: Resultsmentioning

confidence: 99%

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Steinkühler¹,

Peruzzi²,

Krüger³

et al. 2023

Preprint

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Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

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Community science designed ribosomes with beneficial phenotypes

Cited by 5 publications

References 44 publications

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

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

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

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

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