title: The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction.
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.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Fritz
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Interdepartmental Program in Biological Sciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA Northwestern Institute on Complex Systems, Northwestern University, 600 Foster Street, Evanston, IL 60208, USA Institute for Bionanotechnology in Medicine, Northwestern University, 303 E. Superior, Chicago, IL 60611, USA Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208, USA m-jewett@northwestern.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D009876:Operon; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D012335:RNA, Ribosomal