Engineered mRNA–ribosome fusions for facile biosynthesis of selenoproteins

Journal Article · Tue Mar 05 00:00:00 EST 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2321700121· OSTI ID:2318753

Thaenert, Anna ^[1]; Sevostyanova, Anastasia ^[1]; Chung, Christina Z. ^[1]; Vargas-Rodriguez, Oscar ^[1]; Melnikov, Sergey V. ^[2]; Söll, Dieter  ^[3]

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1. Department of Molecular Biophysics &, Biochemistry, Yale University, New Haven, CT 06511
2. Biosciences Institute, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom, Biosciences Institute, Newcastle University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom
3. Department of Molecular Biophysics &, Biochemistry, Yale University, New Haven, CT 06511, Department of Chemistry, Yale University, New Haven, CT 06511

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA–ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins’ active sites. Overall, our work demonstrates the feasibility of creating functional mRNA–rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

• Sponsoring Organization: USDOE
• Grant/Contract Number: FG02-98ER20311
• OSTI ID: 2318753
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 121 Journal Issue: 11; ISSN 0027-8424
• Publisher: Proceedings of the National Academy of Sciences
• Country of Publication: United States
• Language: English