Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Chauhan, Gaurav; Norred, S. Elizabeth; Dabbs, Rosemary M.; Caveney, Patrick M.; George, John K. Vincent; Collier, C. Patrick; Simpson, Michael L.; Abel, Steven M.

doi:10.1021/acssynbio.2c00336

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Journal Article · Wed Oct 19 00:00:00 EDT 2022 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.2c00336· OSTI ID:1965248

Chauhan, Gaurav ^[1]; ^[2]; Dabbs, Rosemary M. ^[2]; Caveney, Patrick M. ^[2]; George, John K. Vincent ^[2]; ^[3]; Simpson, Michael L. ^[2]; ^[1]

Univ. of Tennessee, Knoxville, TN (United States)
Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Cell-free protein synthesis is an important tool for studying gene expression and harnessing it for applications. In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. In this work, we show, using complementary computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed, demonstrating that crowding can be used to differentially organize components of gene expression. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. These results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression in cells.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1965248

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 11 Vol. 11; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

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