title: Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction.
abstract: In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system.
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.
authors.forename: Osman K
authors.initials: OK
authors.lastname: Jamil
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Interdisciplinary Biological Sciences Graduate Program, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Simpson Querrey Institute, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, 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; D005362:Ficoll / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D014176:Protein Biosynthesis; D019163:Reducing Agents; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D019163:Reducing Agents; D005362:Ficoll; D011092:Polyethylene Glycols