Biology subjects

Howard, J. A. L.

Publications and source records attributed to Howard, J. A. L..

3 recordsLinked to original sources

bioRxiv2026

A Bottom-Up Platform for Quantitative Single-Molecule Tracking Through Bacterial Biofilm Mimics

Chronic infections persist in large part thanks to protection that biofilms afford their bacterial creators. The extracellular polymeric substance of biofilms is a hydrated matrix of DNA, polysaccharides, and structural proteins, amongst other components, through which nutrients, signalling molecules, and antimicrobial agents must diffuse to reach the bacteria within. Quantitative measurement of transport on the nanoscale within in vivo biofilms remains challenging due to optical heterogeneity, autofluorescence, active remodelling of biofilms and the ambiguity in trajectory reconstruction during single-particle tracking (SPT). Here, we present a methodological framework for measuring molecular transport in defined minimal extracellular matrix models using quantum dots as fluorescent nanoscale probes imaged with high-speed SlimVar microscopy. To establish conditions in which high-diffusivity particle trajectories can be reliably reconstructed, upper limits to quantum dot concentrations were estimated from Brownian motion. The 99th-percentile inter-frame jump distance was estimated from the three-dimensional Brownian jump distance distribution and used to define a target average nearest neighbour distance, and therefore a per-particle volume, used for calculating a concentration which minimises the probability of trajectory collision during data acquisition. Quantum dot movement was imaged at sub-millisecond frame rates and diffusion coefficients were calculated in a 20% glycerol control and in DNA nanostar hydrogels modelling minimal extracellular matrix scaffolds assembled at 250 M and 500 M. Median diffusion coefficients decreased from 94.9 m2*s-1 in glycerol to 15.9 m2*s-1 and 8.3 m2*s-1 in the 250 M and 500 M hydrogels, respectively. More broadly, this work establishes a workflow for quantitative SPT in minimal biofilm models. Rather than attempting to reproduce the full biological complexity of native biofilms, this approach provides the basis of a modular experimental framework in which individual extracellular matrix components can be incorporated sequentially and their effects on molecular transport quantified.

biophysics↗

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗