Biology subjects

Kapanidis, A. N.

Publications and source records attributed to Kapanidis, A. N..

16 recordsLinked to original sources

bioRxiv2026

Widespread coupling of promoters and terminators of transcription in bacteria

Transcription underpins the expression of genetic information and is viewed as a series of independent initiation and termination events. In bacteria, promoters and terminators of transcription are therefore considered distinct regulatory elements. Here, we challenge this view. Genome-scale analyses reveal that promoters and terminators are extensively coupled. Thus, initiation and termination sites, for adjacent transcription units, frequently overlap. This conserved organisation arises because the DNA sequences, which direct termination, also contribute to promoter function. This couples neighbouring transcription units and generates regulatory interference between RNA polymerases. These findings define a universal mechanism for coordinating transcription across bacterial genomes.

microbiology↗

bioRxiv2025

Structure of the conjugation surface exclusion protein TraT

Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In incompatibility group F (IncF) plasmids surface exclusion is mediated by the outer membrane protein TraT. Here we report the cryoEM structure of the TraT exclusion protein complex from the canonical F plasmid of Escherichia coli. TraT is a hollow homodecamer shaped like a chefs hat. In contrast to most outer membrane proteins, TraT spans the outer membrane using transmembrane helices. We use a novel microscopy-based conjugation assay to probe the effects of directed mutagenesis on TraT. Our analysis provides no support for the idea that TraT has specific interactions with partner proteins. Instead, we infer that TraT is most likely to function by physical interference with conjugation.

microbiology↗

bioRxiv2025

DeepTRACE: Flexible Machine Learning for Analysis and Discovery in Single Molecule Tracks

DeepTRACE is a machine learning tool for analysing long, complex single-molecule tracks in living cells; learning from sequences of molecular events using spatial, temporal, and photometric context. It traces how molecular processes unfold over time and space, incorporating past interactions, subcellular location, and photometric properties. DeepTRACE requires only a few hundred annotated tracks and minutes of CPU training, yet outperforms traditional methods and supports extensive downstream analysis, including the discovery of relationships absent from the training data.

biophysics↗

bioRxiv2025

High-throughput Single-Virion DNA-PAINT Reveals Structural Diversity, Cooperativity and Flexibility during Selective Packaging in Influenza

Influenza A, a negative-sense RNA virus, has a genome that consists of eight single-stranded RNA segments. Influenza co-infections can result in re-assortant viruses that contain gene segments from multiple strains, causing pandemic outbreaks with severe consequences for human health. The outcome of re-assortment is likely influenced by a selective sequence-specific genome packaging mechanism. To uncover the contributions of individual segment pairings to selective packaging, we set out to statistically analyse packaging defects and inter-segment distances in individual A/Puerto Rico/8/34 (H1N1) (PR8) virus particles. To enable such analysis, we developed a multiplexed DNA-PAINT approach capable of assessing the segment stoichiometry of more than 10,000 individual virus particles in one experiment; our approach can also spatially resolve the individual segments inside complete virus particles with a localisation precision of ~10 nm. Our results show the influenza genome can be assembled through multiple pathways in a redundant and cooperative process guided by preferentially interacting segment pairs and aided by synergistic effects that enhance genome assembly, driving it to completion. Our structural evidence indicates that the interaction strength of segment pairs affects the spatial configuration of the gene segments, which appears to be preserved in mature virions. As our method quantified the interactions of whole influenza segments instead of identifying individual sequence-based interactions, our results can serve as a template to quantify the contributions of individual sequence motifs to selective packaging.

microbiology↗

bioRxiv2023

In vivo single-molecule imaging of RecB reveals efficient repair of DNA damage in Escherichia coli

Efficient DNA repair is crucial for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends following a DNA double-strand break (DSB) to initiate repair by homologous recombination. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we perform single-molecule microscopy to investigate DNA repair in real-time in E. coli. We quantify RecB single-molecule mobility and monitor the induction of the DNA damage response (SOS response) in individual cells. We show that RecB binding to broken DNA ends leads to efficient repair without SOS induction. In contrast, in a RecB mutant with modified activities leading to the activation of an alternative repair pathway, repair is less efficient and leads to high SOS induction. Our findings reveal how subtle alterations in RecB activity profoundly impact the efficiency of DNA repair in E. coli.

molecular biology↗

bioRxiv2022

Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG

Bacterial gene expression is highly regulated to allow cells to grow and adapt. Much regulation occurs during transcription elongation, where RNA polymerase (RNAP) extends nascent RNA transcripts aided by global and universally-conserved elongation factor NusG. NusG modulates transcription by inhibiting pausing and backtracking; promoting anti-termination on ribosomal RNA (rrn) operons; coupling transcription with translation on mRNA genes; and stimulating Rho-dependent termination on toxic genes. Despite extensive work on NusG, its functional allocation and spatial distribution in vivo is unknown. Here, we addressed these long-standing questions using single-molecule tracking and super-resolution imaging of NusG in live E. coli cells. We found that, under conditions of moderate growth, NusG is mainly present as a population that associates indirectly with the chromosome via RNAP in transcription elongation complexes, and a slowly diffusing population we identified as a NusG complex with the 30S ribosomal subunit; this complex offers a "30S-guided" path for NusG to enter transcription elongation. Only ~10% of total NusG was fast-diffusing, with the mobility of this population suggesting that free NusG interacts non-specifically with DNA for >50% of the time. Using antibiotics and deletion mutants, we showed that most chromosome-associated NusG is involved in rrn anti-termination and in transcriptiontranslation coupling. NusG involvement in rrn anti-termination was mediated via its participation in phase-separated transcriptional condensates. Our work illuminates the diverse activities of a central regulator while offering a guide on how to dissect the roles of multi-functional machines using in vivo imaging.

molecular biology↗

bioRxiv2022

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

biophysics↗

bioRxiv2022

RNA polymerase redistribution and increased gene dosage support growth in E. coli strains with a minimal number of ribosomal RNA operons

Bacterial transcription by RNA polymerase (RNAP) is spatially organised. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich media, with several studies linking RNAP clustering and transcription of ribosomal RNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons ({Delta}rrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting 5 or 6 of the 7 chromosomal rrn operons. Spatial analysis in{Delta} rrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at the cell end-caps, where the remaining rrn operons reside. These results support a model where RNAPs in{Delta} rrn strains relocate to copies of the remaining rrn operons. We also show that{Delta} rrn strains experience increased rrn gene dosage in rich media, minimising growth defects due to rrn deletions. Our study further links RNAP clusters and rrn transcription, and offers insight on how bacteria maintain growth in the presence of only 1-2 rrn operons.

microbiology↗

bioRxiv2021

High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization

Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. Finally, we showed that in long filaments, viral RNP complexes are located preferentially within Archetti bodies, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.

microbiology↗

bioRxiv2021

The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-{sigma}70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the {beta} rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

biochemistry↗

bioRxiv2020

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Forster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes [~] 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

biophysics↗

bioRxiv2019

Gateway mechanism confers ultrasensitivity on motor’s function

Biological switches are wide spread in many biological systems. Among them, the switch of the bacterial flagellar motor has generated much interest because it affects a mechanical process rather than a chemical reaction, it controls the direction of rotation of a rotary motor rather than being an on/off switch, and it is exceptionally ultrasensitive. Yet, the molecular mechanism underlying its function has remained unknown. Here we resolved unique features of this mechanism: On the one hand, it is tightly regulated by multiple means, involving three binding sites and two different covalent modifications, with the binding specificity being dictated by the type of covalent modification and by a strict binding sequence. On the other hand, it endows the motor with flexibility as it involves an intermediate stage of brief switches that provides a "go/no go" situation, in which the motor can either proceed to a stable rotation in the new direction or shift back to the original direction. This intermediate stage appears to be a means of the cell to produce angular deflection of swimming while maintaining directional persistence. Furthermore, we show by mathematical modeling that such a switching mechanism can provide ultrasensitivity. This unique combination of tight regulation, flexibility, and ultrasensitivity makes this switching mechanism of special interest.

microbiology↗

bioRxiv2019

Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool.

microbiology↗

bioRxiv2019

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilised vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3 terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3 region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication.

microbiology↗

bioRxiv2018

Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120{degrees}) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<{Delta}FRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state ({Delta}G < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.\n\nSignificance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

biophysics↗

bioRxiv2017

Pausing controls branching between productive and non-productive pathways during initial transcription

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region {sigma}3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

biophysics↗