In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

Jewett, Michael C.; Fritz, Brian R.; Timmerman, Laura E.; Church, George M.

doi:10.1038/msb.2013.31

In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

Journal Article · Tue Jun 25 00:00:00 EDT 2013 · Molecular Systems Biology

DOI:https://doi.org/10.1038/msb.2013.31· OSTI ID:1623809

Jewett, Michael C. ^[1]; Fritz, Brian R. ^[2]; Timmerman, Laura E. ^[2]; Church, George M. ^[3]

Harvard Medical School, Boston, MA (United States). Dept. of Genetics; Northwestern Univ., Evanston, IL (United States). Dept. of Chemical and Biological Engineering; DOE/OSTI
Northwestern Univ., Evanston, IL (United States). Dept. of Chemical and Biological Engineering
Harvard Medical School, Boston, MA (United States). Dept. of Genetics

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

View Accepted Manuscript (DOE)

Research Organization:: Northwestern Univ., Evanston, IL (United States)

Sponsoring Organization:: Defense Advanced Research Projects Agency (DARPA); National Institutes of Health (NIH); National Science Foundation (NSF); US Army Research Office (ARO); US Department of the Navy, Office of Naval Research (ONR); USDOE; Wyss Institute for Biologically Inspired Engineering

OSTI ID:: 1623809

Journal Information:: Molecular Systems Biology, Journal Name: Molecular Systems Biology Journal Issue: 1 Vol. 9; ISSN 1744-4292

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
biochemistry & molecular biology
cell-free synthetic biology
in vitro
proteins
ribosome
synthetic biology
transcription
translation

In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

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