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In vitro ribosome synthesis and evolution through ribosome display.

ABSTRACT: 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?×?10⁷ member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4?×?10³ 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.

SUBMITTER: Hammerling MJ

PROVIDER: S-EPMC7048773 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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In vitro ribosome synthesis and evolution through ribosome display.

Hammerling Michael J MJ Fritz Brian R BR Yoesep Danielle J DJ Kim Do Soon DS Carlson Erik D ED Jewett Michael C MC

Nature communications 20200228 1

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<sup>7</ ...[more]

PMID: 32111839

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