Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

Abstract

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

“…The extent of the third, highly mobile fraction, which dominated in the quiescent state, decreased dramatically when cells were serum stimulated. This switch in chromatin mobility is suggestive of dramatically altered molecular crowding, as a result accrued concentration of players of all types in a highly transcribing nucleus, in particular ribosome biogenesis [20,[45][46][47].…”

Controlling Cell-Free Gene Expression Behavior by Tuning Membrane Transport Properties

“…Synthetic biology approaches to controlling gene expression level and variability have focused on genetic circuits. However, the central feature of cell-free synthetic biology is the ability to define the environment by manipulating confinement volume (Caveney et al, 2016), degree of macromolecular crowding (Norred et al, 2018), and the composition of cell extract (Garcia et al, 2018). Likewise, the results reported here show that membrane engineering is a viable approach to control expression behavior and demonstrate a simple optical treatment that greatly diminishes the variability in the permeability of POPC vesicle membranes.…”

“…A broader goal is the realization of more complex cell-free systems (Perez et al, 2016) that may approach cell-like capabilities (Scott et al, 2016). However, these aspirations are stymied by highly variable behavior from identical cell-free expression reactors, especially at cell-relevant reactor volumes (Boreyko et al, 2017;Caveney et al, 2016;Hansen et al, 2015;Norred et al, 2018). Even in simple, single-gene, expression experiments, protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers (Nourian and Danelon, 2013;Saito et al, 2009).…”

Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

“…Perhaps cell and cell-free synthetic biology differ most in the emphasis each place either on gene circuits or on the environment in which gene circuits reside. Cell-free synthetic biology is placing ever greater emphasis on controlling the gene circuit environment by manipulating confinement volume (Caveney et al, 2016), degree of macromolecular crowding (Norred et al, 2018), and the composition of cell extract (Garcia et al, 2018). A defining feature of the gene circuit environment in cells is the controlled transport of molecules across membranes.…”