ArticleNucleic acids research2014
The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction.
Article in Nucleic acids research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 32 papers.
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Who cites it
32 citing papers in PubMed, 60 citations in OpenAlex.
- Evolution of a core ribosomal innovation in octopus.bioRxiv : the preprint server for biology · 2026Article
- Innovations, Challenges and Future Directions of T7RNA Polymerase in Microbial Cell Factories.ACS synthetic biology · 2025Review
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- Autonomous ribosome biogenesis in vitro.Nature communications · 2025Article
- Article
- Assembly of the bacterial ribosome with circularly permuted rRNA.Nucleic acids research · 2024Article
- Alternate conformational trajectories in ribosome translocation.PLoS computational biology · 2024Article
- Critical steps in the assembly process of the bacterial 50S ribosomal subunit.Nucleic acids research · 2024Review
- Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.bioRxiv : the preprint server for biology · 2024Article
- Global profiling of ribosomal protein acetylation reveals essentiality of acetylation homeostasis in maintaining ribosome assembly and function.Nucleic acids research · 2023Article
- Emerging Quantitative Biochemical, Structural, and Biophysical Methods for Studying Ribosome and Protein-RNA Complex Assembly.Biomolecules · 2023Review
- Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.Nucleic acids research · 2023Article
- The Long Road to a Synthetic Self-Replicating Central Dogma.Biochemistry · 2023Review
- Community science designed ribosomes with beneficial phenotypes.Nature communications · 2023Article
- Cell-free Biosynthesis of Peptidomimetics.Biotechnology and bioprocess engineering : BBE · 2023Review
- Computationally-guided design and selection of high performing ribosomal active site mutants.Nucleic acids research · 2022Article
- Engineering Ribosomes to Alleviate Abiotic Stress in Plants: A Perspective.Plants (Basel, Switzerland) · 2022Article
- Article
- Mutational characterization and mapping of the 70S ribosome active site.Nucleic acids research · 2020Article
- In vitro ribosome synthesis and evolution through ribosome display.Nature communications · 2020Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3' modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants.
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