Biology subjects

Brewster, R. C.

Publications and source records attributed to Brewster, R. C..

9 recordsLinked to original sources

bioRxiv2024

E. coli transcription factors regulate promoter activity by a universal, homeostatic mechanism

Transcription factors (TFs) may activate or repress gene expression through an interplay of different mechanisms, including RNA polymerase (RNAP) recruitment, exclusion, and initiation. TFs often have drastically different regulatory behaviors depending on promoter context and interacting cofactors. However, the detailed mechanisms by which each TF affects transcription and produce promoter-dependent regulation is unclear. Here, we discover that a simple model explains the regulatory effects of E. coli TFs in a range of contexts. Specifically, we measure the relationship between basal promoter activity and its regulation by diverse TFs and find that the contextual changes in TF function are determined entirely by the basal strength of the regulated promoter: TFs exert lower fold-change on stronger promoters under a precise inverse scaling. Remarkably, this scaling relationship holds for both activators and repressors, indicating a universal mechanism of gene regulation. Our data, which spans between 100-fold activation to 1000-fold repression, is consistent with a model of regulation driven by stabilization of RNAP at the promoter for every TF. Crucially, this indicates that TFs naturally act to maintain homeostatic expression levels across genetic or environmental perturbations, ensuring robust expression of regulated genes.

systems biology↗

bioRxiv2024

Probing the dependence of transcription factor regulatory modes on promoter features

Transcription Factors (TFs) are often classified as activators or repressors, yet these context-dependent labels are inadequate to predict quantitative profiles that emerge across different promoters. The regulatory interplay between a TFs function and promoter features can be complex due to the lack of systematic genetic control in the natural cellular environment. To address this, we use a library of E. coli strains with precise control of TF copy number. We measure the quantitative regulatory input-output function of 90 TFs on synthetic promoters that isolate the contributions of TF binding sequence, location, and basal promoter strength to gene expression, uncovering TF specific regulatory principles. We infer that many of these TFs function by stabilizing RNA polymerase at the promoter, a property we see for both activating and repressing TFs. We develop a thermodynamic model that predicts stabilizing TFs have a specific quantitative relationship with promoters of differential strength. We test this prediction using synthetic promoters spanning over 100-fold range in basal expression levels and confirm that stronger promoters have lower fold-change for stabilizing TFs, whereas non-stabilizing TFs do not exhibit this relationship, indicating a conserved mechanism of transcription control across distinct TFs. This work demonstrates that understanding the intrinsic mechanisms of TF function is central to decoding the relationship between sequence and gene expression.

systems biology↗

bioRxiv2023

Regulatory properties of transcription factors with diverse mechanistic function

Transcription factors (TFs) regulate the process of transcription through the modulation of different kinetic steps. Although models can often describe the observed transcriptional output of a measured gene, predicting a TFs role on a given promoter requires an understanding of how the TF alters each step of the transcription process. In this work, we use a simple model of transcription to assess the role of promoter identity, and the degree to which TFs alter binding of RNAP (stabilization) and initiation of transcription (acceleration) on three primary characteristics: the range of steady-state regulation, cell-to-cell variability in expression, and the dynamic response time of a regulated gene. We find that steady state regulation and the response time of a gene behave uniquely for TFs that regulate incoherently, i.e that speed up one step but slow the other. These TFs function as activators when regulating weak promoters but switch to repression when the promoters are strong or vice versa. Furthermore, we find that TFs with this regulatory make-up have dynamic implications, with one type of incoherent mode configuring the promoter to respond more slowly at intermediate TF concentrations. We also demonstrate that the noise of gene expression for these TFs is sensitive to promoter strength, with a distinct non-monotonic profile that is apparent under stronger promoters. Taken together, our work uncovers the coupling between promoters and TF regulatory modes with implications for understanding natural promoters and engineering synthetic gene circuits with desired expression properties.

systems biology↗

bioRxiv2022

Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli

The fundamental biology and application of bacterial exopolysaccharides is gaining increasing attention. However, current synthetic biology efforts to produce the major component of Escherichia sp. slime, colanic acid, and functional derivatives thereof have been limited. Herein, we report the overproduction of colanic acid (up to 1.32 g/L) from D-glucose in an engineered strain of E. coli JM109. Furthermore, we report that chemically-synthesized L-fucose analogues containing an azide motif can be metabolically incorporated into the slime layer via a heterologous fucose salvage pathway from Bacteroides sp. and used in a click reaction to attach an organic cargo to the cell surface. This molecular engineered bio-polymer possesses enormous potential as a new tool for use in chemical, biological and materials research.

bioengineering↗

bioRxiv2021

Tunable Transcription Factor Library for Robust Quantification of Gene Expression Dynamics in E. coli

Predicting the quantitative regulatory function of a TF based on factors such as binding sequence, binding location and promoter type is not possible. The interconnected nature of gene networks and the difficulty in tuning individual TF concentrations makes the isolated study of TF function challenging. Here we present a library of E. coli strains designed to allow for precise control of the concentration of individual TFs enabling the study of the role of TF concentration on physiology and regulation. We demonstrate the usefulness of this resource by measuring the regulatory function of the zinc responsive TF, ZntR and the paralogous TF pair, GalR/GalS. For ZntR, we find that zinc alters ZntR regulatory function in a way that enables activation of the regulated gene to be robust with respect to ZntR concentration. For GalR and GalS, we are able to demonstrate that these parlogous TFs have fundamentally distinct regulatory roles beyond differences in binding affinity.

systems biology↗

bioRxiv2021

Controlling gene expression timing through gene regulatory architecture

Gene networks typically involve the regulatory control of multiple genes with related function. This connectivity enables correlated control of the levels and timing of gene expression. Here we study how gene expression timing in the single-input module motif can be encoded in the regulatory DNA of a gene. Using stochastic simulations, we examine the role of binding affinity, TF regulatory function and network size in controlling the mean first-passage time to reach a fixed fraction of steady-state expression for both an auto-regulated TF gene and a target gene. We also examine how the variability in first-passage time depends on these factors. We find that both network size and binding affinity can dramatically speed up or slow down the response time of network genes, in some cases predicting more than a 100-fold change compared to that for a constitutive gene. Furthermore, these factors can also significantly impact the fidelity of this response. Importantly, these effects do not occur at "extremes" of network size or binding affinity, but rather in an intermediate window of either quantity. Author summaryRegulated genes are able to respond to stimuli in order to ramp up or down production of specific proteins. Although there is considerable focus on the magnitude (or fold-change) of the response and how that depends on the architectural details of the regulatory DNA, the dynamics, which dictates the response time of the gene, is another key feature of a gene that is encoded within the DNA. Unraveling the rules that dictate both the response time of a gene and the precision of that response encoded in the DNA poses a fundamental problem. In this manuscript, we systematically investigate how the response time of genes in auto-regulatory networks is controlled by the molecular details of the network. In particular, we find that network size and TF-binding affinity are key parameters that can slow, in the case of auto-activation, or speed up, in the case of auto-repression, the response time of not only the auto-regulated gene but also the genes that are controlled by the auto-regulated TF. In addition, we find that the precision of the response depends crucially on these characteristics.

systems biology↗

bioRxiv2021

Quantifying the regulatory role of individual transcription factors in Escherichia coli

Gene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, with a net regulation that depends on both the direct interactions of TFs with RNA polymerase (RNAP) and the indirect interactions with each other. Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate the individual TFs regulatory effect. Using a thermodynamic model, the direct regulatory impact of the TF on RNAP is decoupled from TF occupancy and interpreted as acting through two mechanisms: (de)stabilization of RNAP and (de)acceleration of transcription initiation. We find the contributions of each mechanism depends on TF identity and binding location; for the set of TFs profiled, regulation immediately downstream of the promoter is insensitive to TF identity, yet these same TFs regulate by distinct mechanisms upstream of the promoter. Strikingly, we observe two fundamental regulatory paradigms with these two mechanisms acting coherently, to rein-force the observed regulatory role (activation or repression), or incoherently, where the TF regulates two distinct steps with opposing effect. This insight provides critical information on the scope of TF-RNAP regulation allowing for a stronger approach to characterize the endogenous regulatory function of TFs.

systems biology↗

bioRxiv2019

Regulatory asymmetry in the negative single-input module network motif: Role of network size, growth rate and binding affinity

The single-input module (SIM) is a regulatory motif capable of coordinating gene expression across functionally related genes. We explore the relationship between regulation of the central autoregulated TF in a negatively regulated SIM and the target genes using a synthetic biology approach paired with stochastic simulations. Surprisingly, we find a fundamental asymmetry in the level of regulation experienced by the TF gene and its targets, even if they have identical regulatory DNA; the TF gene experiences stronger repression than its targets. This asymmetry is not predicted from deterministic modeling of the system but is revealed from corresponding stochastic simulations. The magnitude of asymmetry depends on factors such as the number of targets in the SIM, TF degradation rate (or growth rate) and TF binding affinity. Beyond implications for SIM motifs, the influence of network connectivity on regulatory levels highlights an interesting challenge for predictive models of gene regulation.

systems biology↗

bioRxiv2018

A Persistence Detector for Metabolic Network Rewiring in an Animal

Biological systems must possess mechanisms that prevent inappropriate responses to spurious environmental signals. Gene regulatory network circuitries known as coherent type 1 feed-forward loops (FFLs) with AND-logic gates have been proposed to function as a persistence detector because it generates a delay in target activation and prevents target induction unless the input signal is sustained. While such a circuit has been found for the L-arabinose utilization system in E. coli, their existence and relevance multicellular organisms has remained unclear. Here, we identify the first persistence detector in an animal that redirects propionate breakdown to a shunt pathway when flux through the canonical propionate breakdown pathway is perturbed. We propose that this mechanism has evolved to ensure the shunt pathway stays off unless propionate accumulation is persistent because the shunt pathway generates highly toxic acrylate. Our study uniquely connects persistence detector circuitry to a physiological response in an animal.

systems biology↗