Biology subjects

Duss, O.

Publications and source records attributed to Duss, O..

7 recordsLinked to original sources

bioRxiv2026

A bacterial nucleolus for cold adaptation

Compartmentalisation enables cells to spatially organise essential biochemical processes. This is exemplified by the eukaryotic nucleolus, which concentrates the machinery for ribosome biogenesis. Bacteria are generally thought to lack an equivalent compartment. Here we discover a nucleolus-like condensate in Escherichia coli that emerges at rDNA loci during cold adaptation to spatially organise and promote ribosome biogenesis. Low temperatures, which stabilise RNA secondary structures, induce accumulation of the RNA-remodelling enzyme CsdA. This raises the cellular CsdA concentration above its condensation threshold, driving condensate formation at rDNA loci when rRNA transcription is active. CsdA compartments enrich the rRNA transcription machinery and ribosome-biogenesis factors, spatially linking rRNA synthesis to downstream processing and assembly. Disrupting CsdA condensation impairs ribosome maturation and reduces bacterial adaptation to cold temperature, demonstrating functional importance of condensate formation. Comparative genomics suggests that analogous condensate-forming RNA remodelling enzymes have evolved repeatedly across diverse bacterial lineages. Our findings establish biomolecular condensates as a unifying organisational principle of ribosome biogenesis across the tree of life and suggest that adaptive compartmentalisation can emerge when environmental conditions challenge the efficiency of essential biochemical processes.

cell biology↗

bioRxiv2026

Functional coupling between ribosomal RNA transcription and processing guided by stable transcription factor binding

Coordinating ribosomal RNA (rRNA) transcription, folding and processing is essential for bacterial ribosome assembly. Yet, the molecular mechanisms underlying this coordination remain poorly understood. Particularly, whether the rRNA transcription antitermination complex (rrnTAC: NusA, NusG, NusB, NusE, S4, SuhB) orchestrates this coordination remains unclear. Here, we develop a suite of multi-color single-molecule fluorescence microscopy assays to simultaneously visualize, in real-time, rrnTAC assembly dynamics and their effect on transcription and co-transcriptional processing by RNase III. We find that transient ([~]1 s) interactions of general transcription factors NusA and NusG with RNAP, and of NusB/E with the rRNA-specific boxBAC element, are stabilized to minutes-long residence by final SuhB recruitment. Stable rrnTAC assembly is required to reduce RNAP pausing and to boost co-transcriptional rRNA processing. Collectively, our results reveal how transcription factor binding dynamics relate to function: transient for messenger RNA transcription and stable for rRNA transcription and processing.

biophysics↗

bioRxiv2025

Functional characterization of dynamic nascent RNA folding ensembles in real-time

RNA structure starts forming co-transcriptionally as the nascent RNA emerges from the RNA polymerase and is dynamically modulated by cellular factors. How individual RNA conformations, out of an ensemble of RNA molecules, relate to function is not well understood. Here, developing multi-color single-molecule fluorescence microscopy experiments, we track in real-time nascent RNA structure formation, functionally characterizing up to 8 different types of RNA molecules. We find that ribosomal proteins, RNA modification enzymes or antisense oligonucleotides specifically modulate a subset of the RNA folding classes. For example, we provide direct evidence that increased local RNA accessibility at specific sites correlates with the chaperoning activity of ribosomal proteins during ribosome assembly. These experiments provide a general framework to study how dynamic RNA folding, and misfolding, relates to function.

biophysics↗

bioRxiv2025

Real-time tracking of mRNP complex assembly reveals various mechanisms that synergistically enhance translation repression

Protein biosynthesis must be highly regulated to ensure proper spatiotemporal gene expression and thus cellular viability. Translation is often modulated at the initiation stage by RNA binding proteins through either promotion or repression of ribosome recruitment to the mRNA. However, it largely remains unknown how the kinetics of mRNA ribonucleoprotein (mRNP) assembly on untranslated regions (UTRs) relates to its translation regulation activity. Using Sex-lethal (Sxl)-mediated translation repression of msl-2 in female fly dosage compensation as a model system, we show that different mechanisms in mRNP assembly synergistically achieve tight translation repression. Using multi-color single-molecule fluorescence microscopy we show that 1) Sxl targets its binding sites via sliding and double-binding, 2) that Unr recruitment is accelerated over 500-fold by RNA-bound Sxl and 3) that Hrp48 further stabilizes RNA-bound Sxl indirectly via ATP-independent RNA remodeling. Overall, we provide a framework to study how multiple RBPs dynamically cooperate with RNA to achieve function.

biophysics↗

bioRxiv2025

Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

Influenza A virus (IAV) hijacks host cellular machinery, but many virus-IAV interactions and contacting protein sites remain uncharacterised, particularly those dependent on intact cellular architecture, such as membrane-associated or phase-separated compartments. Here, we applied in-cell cross-linking mass spectrometry (XL-MS), integrated with AlphaFold-based structural modelling and functional assays, to map protein-protein contact sites in IAV-infected human cells. This approach revealed previously unrecognised virus-host interactions linked to spatially organised processes, including the maturation pathway of HA through the membrane-bound ER- Golgi system, the novel interaction of M2 with the membrane-embedded LAT1 amino acid transporter, and the progressive disassembly of paraspeckles-phase-separated compartments in the nucleus. We validate M2-LAT1 interaction and paraspeckle disassembly in human primary lung epithelial cells and show that the paraspeckle disassembly constitutes a new and unique infection mechanism through which IAV releases RNA-binding proteins that support viral RNA replication. These findings advance the understanding of IAV manipulation of host cellular processes and illustrate how the integrative in-cell structural system biology approach captures native host-pathogen interactomes, infection pathways, and host cell perturbations.

microbiology↗

bioRxiv2024

Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC

Transcription of tRNA genes by RNA polymerase III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryo-EM and single-molecule FRET (smFRET) to reveal structural and real-time kinetic insights into how the 520 kDa yeast TFIIIC complex engages A- and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of {tau}A and {tau}B subcomplexes bound to the A- and B-box were obtained at 3.7 and 2.5 [A] resolution, respectively, while cryo-EM single particle mapping determined the specific distance and relative orientation of the {tau}A and {tau}B subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of {tau}A and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

biochemistry↗

bioRxiv2023

Tracking transcription-translation coupling in real-time

A central question in biology is how macromolecular machines function cooperatively. In bacteria, transcription and translation occur in the same cellular compartment and can be physically and functionally coupled. While several recently published high-resolution structures of the ribosome-RNA polymerase (RNAP) complex provided first mechanistic insight into the coupling process, we do not know how these structural snapshots are placed along a dynamic reaction trajectory. Here, we reconstitute a complete active transcription-translation system and develop multi-color single-molecule fluorescence microscopy experiments to directly and simultaneously track transcription elongation, translation elongation and the physical and functional coupling between the ribosome and the RNAP in real-time. Our data show that the ribosome slows down while colliding with the RNAP and that coupling following a collision becomes less efficient. Unexpectedly, physical coupling can occur with hundreds of nucleotides of intervening mRNA between both machineries by mRNA looping, and increases in efficiency in presence of NusG. We detect active transcription elongation during mRNA looping and show that NusA-paused RNAPs can be activated by the ribosome by long-range physical coupling. We provide an alternative explanation on how the ribosome can rescue RNAP from frequent pausing without requiring collisions by a closely trailing ribosome. Our data mechanistically highlight an example of how macromolecular machines central to gene expression physically and functionally cooperate.

biophysics↗