Biology subjects

Khamis, H.

Publications and source records attributed to Khamis, H..

10 recordsLinked to original sources

bioRxiv2026

Tunable kinetic destabilization governs RNA polymerase passage through a DNA-bound transcription factor

Eukaryotic transcription factors recognize short motifs, creating abundant binding sites within gene bodies and potential collisions with elongating RNA polymerases, yet how such encounters are resolved remains unclear. Here, we use optical tweezers to monitor RNA polymerase transcription through DNA-bound Egr-1, a zinc-finger transcription factor. Using DNA-fluctuation suppression as a readout of polymerase arrival, we show that Egr-1 delays elongation in an orientation- and rNTP-dependent manner, whereas force measurements indicate that RNAP does not bypass the TF by mechanical eviction. Instead, RNAP destabilizes the Egr-1-DNA complex over a short, structured interaction zone, increasing TF dissociation non-monotonically with distance. Monte Carlo simulations incorporating these kinetic changes recapitulate passage-time distributions. CpG methylation shortens Egr-1 residence time and largely eliminates the TF-dependent delay, suggesting a role for gene-body methylation in reducing kinetic barriers to elongation. These results reveal DNA-bound TFs as tunable barriers that locally shape transcription elongation.

biophysics↗

bioRxiv2025

Intrinsically disordered regions facilitate Msn2 target search to drive promoter selectivity

Transcription factors (TFs) regulate gene expression by binding specific DNA motifs, yet only a fraction of putative sites is occupied in vivo. Intrinsically disordered regions (IDRs) have emerged as key contributors to promoter selectivity, but the underlying mechanisms remain incompletely understood. Here, we use single-molecule optical tweezers to dissect how IDRs influence DNA binding by Msn2, a yeast stress-response regulator. We show that IDRs facilitate initial non-specific association with DNA and promote one-dimensional diffusion toward target motifs, supported by charge-mediated interactions. Remarkably, the IDR-dependent search mechanism displays sequence sensitivity, with promoter-derived sequences enhancing both initial binding and sliding rates, demonstrating that Msn2-DNA interactions alone are sufficient to confer promoter selectivity in the absence of chromatin or cofactors. These findings provide direct mechanistic evidence for how IDRs tune transcription factor search dynamics and expand sequence recognition beyond canonical motifs, supporting a mechanism for promoter selectivity in complex genomic contexts.

biophysics↗

bioRxiv2025

Optimizing DNA origami assembly through selection of scaffold sequences that minimise off-target interactions

DNA origami is a mainstay of DNA nanotechnology and several efforts have been devoted to understanding how various factors of the self-assembly reaction affect the final yield of the target origami structure. This study analyses how base sequence affects origami yield through the generation of off-target side reactions during selfassembly. Off-target bindings are an under-explored phenomenon and can potentially introduce unwanted assembly barriers and kinetic traps in the origami folding pathway. We developed a multi-objective computational approach that takes a given origami design and scores different scaffold sequences (and their complementary staples) for the prevalence of four different types of off-target binding events. Using our method on DNA origami, we can select bad regions of biological sequences (like lambda DNA phage) that, when used as origami scaffold sequences, have an excessive number of off-target side reactions for each shape. We show, using high-resolution atomic force microscopy (AFM), that these scaffold sequences largely fail to fold into the target triangle or rectangle structure in vitro, despite the scaffold sequence having a fully complementary staple set present. Conversely, using our method we can also select good regions of biological sequences. These sequences are deficient in off-target reactions and when used as origami scaffolds, fold more successfully into their target structures as characterised by AFM. These results have been validated in "blind" folding experiments at two different laboratories in which the experimenters did not know which scaffolds were good or bad folders. To further investigate assembly behaviour, optical tweezers experiments revealed distinct mechanical response profiles, correlating with scaffold-specific off-target interactions. While variants with higher GC content show a high mean unfolding force, variants with lower off-target binding demonstrated more uniform force-extension curves. Our analysis confirmed that high off-target binding leads to increased structural heterogeneity, as seen in the clustering behaviour of unfolding traces of OT experiments. Over-all, our work demonstrates how the off-target reactions implicit in base sequences can derail the origami self-assembly process if sufficiently prevalent, and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design.

bioengineering↗

bioRxiv2023

Role of finger movement kinematics in friction perception at initial contact with smooth surfaces

When manipulating objects, humans adjust grip force to friction remarkably quickly: it may take just 100 ms to see adjustment to friction at the skin-object interface. While the motor commands adapt, subjects become aware of slipperiness of touched surfaces. In this study, we explore the sensory processes underlying such friction perception when no intentional exploratory sliding movements are present. Previously, we have demonstrated that humans cannot perceive frictional differences when surfaces are brought in contact with an immobilized finger (Khamis et al., 2021b) unless there is a submillimeter lateral displacement (Afzal et al., 2022), or subjects made the movement themselves (Willemet et al., 2021). In the current study, subjects actively interacted with a device that can modulate friction using ultrasound, without an exploratory sliding movement, as they would when gripping an object to lift it. Using a two-alternative forced-choice paradigm, subjects had to indicate which of two surfaces felt more slippery. Subjects could correctly identify the more slippery surface in 87 {+/-} 8% of cases (mean{+/-}SD; n=12). Biomechanical analysis of finger pad skin contacting a flat smooth surface indicated that natural movement kinematics (e.g., tangential movement jitter and physiological tremor) may enhance perception of frictional effects. To test whether this is the case, in a second experiment a hand support was introduced to limit fingertip movement deviation from a straight path. Subject performance significantly decreased (66 {+/-} 12% correct, mean{+/-}SD; n=12), indicating that friction perception at the initial contact is enhanced or enabled by natural movement kinematics. Significance statementSensing surface friction is crucial for automatic grip force control to avoid dropping objects. A slipping handhold can lead to loss of balance and falling. In many instances, the required grip force may exceed hands physical ability or an objects breakage point, therefore cognitive selection of a safe and achievable action plan based on friction perception is critical. Little is known about how our awareness of surface slipperiness is obtained under such circumstances without exploratory movement. The current study demonstrates that natural movement kinematics inducing submillimeter lateral movements play a central enabling role, demonstrating interdependence between the motor system and sensory mechanisms. These findings broaden our fundamental understanding of sensorimotor control and could inform the development of advanced sensor technologies.

neuroscience↗

bioRxiv2022

How tactile afferents in the human fingerpad encode tangential torques associated with manipulation: are we better than monkeys?

Dexterous object manipulation depends critically on information about forces normal and tangential to the fingerpads, and also on torque associated with object orientation at grip surfaces. In this study we investigated how torque information is encoded by human tactile afferents, including slowly adapting type-II (SA-II) afferents, in the fingerpads. SA-II afferent properties seemed perfectly suited for torque encoding but could not be previously investigated as they are absent in the glabrous skin of monkeys. Torques of different magnitudes (3.5-7.5 mNm) were applied in clockwise and anticlockwise directions to a standard central site on the fingerpads of 34 participants. Torques were superimposed on a 2 N, 3 N, or 4 N background normal force. Unitary microneurography recordings were made from fast adapting type-I (FA-I, n=39), slowly adapting type-I (SA-I, n=31), and type-II (SA-II, n=13) afferents supplying the fingerpads. All three afferent types encoded torque magnitude and direction, with SA-II afferents showing excitatory and inhibitory modulation depending on torque direction. Most afferents of all three types had higher torque sensitivity with smaller normal force. FA-I afferents showed the best torque magnitude and worst directional discrimination abilities in both species. Human SA-I afferent response to static torque was inferior to dynamic stimuli, while in monkeys the opposite was true. In humans this might be compensated by the addition of a sustained SA-II afferent input. In comparison to monkeys the performance of each afferent type was inferior in humans, likely due to differences in fingertip tissue compliance and skin friction. [currently 246 words; 250 words max] Significance StatementWe investigate how individual human tactile nerve fibres encode rotational forces (torques) and compare them to their monkey counterparts. Human hands, but not monkey hands, are innervated by a tactile neuron type (SA-II afferents) specialised to encode directional skin strain, yet so far, torque encoding has only been studied in monkeys. We find that human SA- I afferents were generally less sensitive and less able to discriminate torque magnitude and direction than their monkey counterparts, especially during the static phase of torque loading. However, this shortfall in humans could be compensated by the addition of SA-II afferent input. This indicates that variation in afferent types might compliment their specialisation for stimulus features, by slight differences between encoding in the two species.

neuroscience↗

bioRxiv2021

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a proteins binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

biophysics↗

bioRxiv2020

Extended and dynamic linker histone-DNA interactions control chromatosome compaction

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as ~ {+/-}140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosomes entry and exit. These interactions are dynamic, exhibiting rapid binding and dissociation, sensitive to phosphorylation of a specific residue, and crucial to determining the symmetry of the chromatosomes core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

biophysics↗

bioRxiv2017

The mechano-chemistry of reverse transcriptase

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

biophysics↗

bioRxiv2017

Single-molecule DNA unzipping reveals asymmetric modulation of the transcription factor Egr-1 by its binding site sequence and context

Most functional transcription factor (TF) binding sites deviate from their \"consensus\" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

biophysics↗

bioRxiv2017

Mobile nucleosomes and the accessibility of transcription factors to DNA: a modified dynamic equilibrium model

Nucleosomes, the basic building block of chromatin, regulate the accessibility of the transcription machinery to DNA. Recent studies have revealed that the nucleosome's spontaneous, thermally driven positional dynamics are modulated by different factors, and exploited by the cell as a regulatory mechanism. In particular, enrichment of mobile nucleosomes at the promoters of genes suggests that the mobility of nucleosomes may affect the ability of transcription factors to bind DNA. However, a quantitative model describing the effect nucleosome mobility on the effective affinity of transcription factors is lacking. We present here a simple equilibrium model that captures the essence of the effect, and show that modulation of the nucleosome's mobility can be a potent and versatile regulator of transcription factor binding.

biophysics↗