Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 29; pp. 11692 - 11697
• Main Authors: Sokolova, Ekaterina, Spruijt, Evan, Hansen, Maike M. K., Dubuc, Emilien, Groen, Joost, Chokkalingam, Venkatachalam, Piruska, Aigars, Heus, Hans A., Huck, Wilhelm T. S.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 16.07.2013; National Acad Sciences
• Subjects: Artificial Cells; Biological Sciences; Deoxyribonucleic acid; DNA; E coli; Escherichia coli; Fluorescence; Gene expression; Hydrophobic and Hydrophilic Interactions; Kinetics; Macromolecular Substances - chemistry; Messenger RNA; Microscopy, Fluorescence; Models, Biological; Molecules; Phase Transition; Phase transitions; Physical Sciences; Protein synthesis; Proteins; RNA; RNA polymerase; Salts; Transcription, Genetic - physiology; macromolecular crowding; microdroplets
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1222321110

Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.