ArticleNucleic acids research2010
In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme.
Article in Nucleic acids research, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 57 citations in OpenAlex.
- Development of a Microdroplet-Based Functional Genomic Screening Pipeline by Combination of DNA Nanoflowers and PURExpress Cell-Free Expression.ACS synthetic biology · 2026Article
- Gene circuit-based sensors.Fundamental research · 2025Review
- A mathematical model of cell-free transcription-translation with plasmid crosstalk.Synthetic biology (Oxford, England) · 2025Article
- Bacterial chromatin proteins, transcription, and DNA topology: Inseparable partners in the control of gene expression.Molecular microbiology · 2024Review
- Supramolecular protein assembly in cell-free protein synthesis system.Bioresources and bioprocessing · 2022Article
- Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry.Nature nanotechnology · 2020Article
- Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system.Chemical science · 2019Article
- Synthetic Biology Goes Cell-Free.BMC biology · 2019Review
- Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro.Scientific reports · 2019Article
- Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.Journal of the American Chemical Society · 2019Article
- Perspective: Solidifying the impact of cell-free synthetic biology through lyophilization.Biochemical engineering journal · 2018Article
- Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile.Nucleic acids research · 2017Article
- Cell-Free Synthetic Biology: Engineering Beyond the Cell.Cold Spring Harbor perspectives in biology · 2016Review
- DNA Nanoparticles for Improved Protein Synthesis In Vitro.Angewandte Chemie (International ed. in English) · 2016Article
- One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil-water interfaces.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Article
- The PURE system for the cell-free synthesis of membrane proteins.Nature protocols · 2015Article
- High-throughput preparation methods of crude extract for robust cell-free protein synthesis.Scientific reports · 2015Article
- Engineering bacterial transcription regulation to create a synthetic in vitro two-hybrid system for protein interaction assays.Journal of the American Chemical Society · 2014Article
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2 authors at 1 institution in 1 country.
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Abstract
In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes.
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