Biology subjects

Stewart, F.

Publications and source records attributed to Stewart, F..

5 recordsLinked to original sources

bioRxiv2026

Bacterial RNA polymerase exemplifies a general physical mechanism for accelerating protein-DNA association

Bacterial RNA polymerases (RNAPs) have two flexibly tethered subunit C-terminal domains (-CTDs) that bind DNA. Interaction between -CTDs and some promoter DNA motifs is known to accelerate transcription initiation, but the physical mechanism by which it does so is unclear. We used single-molecule multiwavelength fluorescence microscopy to test how the diffusion-limited binding kinetics of core RNAP to non-promoter DNA differ from those of mutant RNAPs that lack one or both -CTDs. We find that even though -CTDs and their tethers are small compared to the complete RNAP molecule, the presence of two -CTDs accelerates DNA binding by [~]10-fold and [~]55-fold respectively relative to RNAP constructs in which one or both -CTDs are deleted. In contrast, the presence of -CTDs did not have a detectable effect on RNAP-DNA complex lifetimes in the absence of RNA synthesis. We explain how -CTDs achieve the dramatic acceleration of RNAP binding to DNA using a quantitative three-state kinetic model that includes a transient binding intermediate where only the -CTD(s) are bound to DNA, tethering the rest of the RNAP in the vicinity of DNA. The model and assumed parameters are validated using Brownian dynamics simulations of the DNA association reactions for two-, one-, or zero-CTD RNAP constructs. The combination of single-molecule experiments, mathematical theory, and simulations suggests that adding a flexible DNA-binding tether is a general physical mechanism which can accelerate the diffusion-limited binding of a large protein like RNAP to DNA and quantitatively defines the conditions under which this acceleration can occur. Significance StatementLarge enzymes that must associate with DNA to perform their biological functions are expected to bind DNA only slowly because of slow enzyme diffusion. However, some DNA-binding enzymes have one or more additional small DNA-binding domains that can diffuse rapidly but are attached to the enzyme through an unstructured flexible tether. Combining single-molecule experiments, theory, and computation, we present evidence for a general mechanism by which such tethered domains can accelerate enzyme binding to DNA by orders of magnitude, while not significantly changing the duration of the DNA-bound state. We demonstrate this effect for the flexibly-tethered -subunit C-terminal domain of bacterial RNA polymerase. The mechanism may allow the polymerase to rapidly bind DNA while minimizing non-functional sequestration on the genome.

biophysics↗

bioRxiv2023

Many morphs: parsing gesture signals from the noise

Parsing signals from noise is a general problem for signallers and recipients, as well as for researchers studying communicative systems. Substantial research efforts have been invested in comparing how other species encode information and meaning in their signals, and how signalling is structured. However, our ability to do so depends on identifying and discriminating signals that represent meaningful units of analysis. Early approaches to defining signal repertoires applied top- down approaches, classifying cases into predefined signal types. Recently, more labour-intensive methods have taken a bottom-up approach describing the features of each signal in detail and clustering cases into types based on patterns of similarity between them in multi-dimensional feature-space that were previously undetectable. Nevertheless, it remains essential to assess whether the resulting repertoires are composed of relevant units from the perspective of the species using them, and redefining repertoires when additional data makes more detailed analyses feasible. In this paper we provide a framework that takes data from the largest set of wild chimpanzee (Pan troglodytes) gestures currently available, splitting gesture types at a fine scale based on modifying features of gesture expression and then determining whether this splitting process increases the information content of the communication system. Our method allows different features of interest to be incorporated into the splitting process, providing substantial future flexibility across - for example - species, populations, and levels of signal granularity. In doing so we provide a powerful tool allowing researchers interested in gestural communication to establish repertoires of relevant units for subsequent analyses within and between systems of communication.

animal behavior and cognition↗

bioRxiv2020

SETD1B controls cognitive function via cell type specific regulation of neuronal identity genes

Histone-3-lysine-4-methylation (H3K4me) is mediated by six different lysine methyltransferases (KMTs). Amongst these enzymes SET domain containing 1b (SETD1B) has been linked to intellectual disability but its role in the adult brain has not been studied yet. Here we show that mice lacking Setd1b from excitatory neurons of the adult forebrain exhibit severe memory impairment. By combining neuron-specific ChIP-seq, RNA-seq and single cell RNA-seq approaches we show that Setd1b controls the expression of neuronal-identity genes with a broad H3K4me3 peak linked to learning and memory processes. Our data furthermore suggest that basal neuronal gene-expression is ensured by other H3K4 KMTs such as Kmt2a and Kmt2b while the additional presence of Setd1b at the single cell level provides transcriptional consistency to the expression of genes important for learning & memory.

neuroscience↗

bioRxiv2020

Ultrasound Mediated Delivery of Quantum Dots from a Capsule Endoscope to the Gastrointestinal Wall

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in-vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focussed ultrasound. These findings suggest that the use of focused ultrasound together with microbubbles could play a role in the oral delivery of biologic therapeutics.

bioengineering↗

bioRxiv2018

Spatial Mark-Resight for Categorically MarkedPopulations with an Application to GeneticCapture-Recapture

The estimation of animal population density is a fundamental goal in wildlife ecology and management, commonly met using mark recapture or spatial mark recapture (SCR) study designs and statistical methods. Mark-recapture methods require the identification of individuals; however, for many species and sampling methods, particularly noninvasive methods, no individuals or only a subset of individuals are individually identifiable. The unmarked SCR model, theoretically, can estimate the density of unmarked populations; however, it produces biased and imprecise density estimates in many sampling scenarios typically encountered. Spatial mark-resight (SMR) models extend the unmarked SCR model in three ways: 1) by introducing a subset of individuals that are marked and individually identifiable, 2) introducing the possibility of individual-linked telemetry data, and 3) introducing the possibility that the capture-recapture data from the survey used to deploy the marks can be used in a joint model, all improving the reliability of density estimates. The categorical spatial partial identity model (SPIM) improves the reliability of density estimates over unmarked SCR along another dimension, by adding categorical identity covariates that improve the probabilistic association of the latent identity samples. Here, we combine these two models into a \"categorical SMR\" model to exploit the benefits of both models simultaneously. We demonstrate using simulations that SMR alone can produce biased and imprecise density estimates with sparse data and/or when few individuals are marked. Then, using a fisher (Pekania pennanti) genetic capture-recapture data set, we show how categorical identity covariates, marked individuals, telemetry data, and jointly modeling the capture survey used to deploy marks with the resighting survey all combine to improve inference over the unmarked SCR model. As previously seen in an application of the categorical SPIM to a real-world data set, the fisher data set demonstrates that individual heterogeneity in detection function parameters, especially the spatial scale parameter{sigma} , introduces positive bias into latent identity SCR models (e.g., unmarked SCR, SMR), but the categorical SMR model provides more tools to reduce this positive bias than SMR or the categorical SPIM alone. We introduce the possibility of detection functions that vary by identity category level, which will remove individual heterogeneity in detection function parameters than is explained by categorical covariates, such as individual sex. Finally, we provide efficient SMR algorithms that accommodate all SMR sample types, interspersed marking and sighting periods, and any number of identity covariates using the 2-dimensional individual by trap data in conjunction with precomputed constraint matrices, rather than the 3-dimensional individual by trap by occasion data used in SMR algorithms to date.

ecology↗