The control of proteins by a transcription factor with periodically varying concentration exhibits intriguing dynamical behaviour. Even though it is accepted that transcription factors vary their dynamics in response to different situations, insight into how this affects downstream genes is lacking. Here, we investigate how oscillations and chaotic dynamics in the transcription factor NF-κB can affect downstream protein production. We describe how it is possible to control the effective dynamics of the transcription factor by stimulating it with an oscillating ligand. We find that chaotic dynamics modulates gene expression and up-regulates certain families of low-affinity genes, even in the presence of extrinsic and intrinsic noise. Furthermore, this leads to an increase in the production of protein complexes and the efficiency of their assembly. Finally, we show how chaotic dynamics creates a heterogeneous population of cell states, and describe how this can be beneficial in multi-toxic environments.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

SUMMARYOscillations and noise drive many processes in biology, but how both affect the activity of the transcription factor nuclear factor κB (NF-κB) is not understood. Here, we observe that when NF-κB oscillations are entrained by periodic tumor necrosis factor (TNF) inputs in experiments, NF-κB exhibits jumps between frequency modes, a phenomenon we call “cellular mode-hopping.” By comparing stochastic simulations of NF-κB oscillations to deterministic simulations conducted inside and outside the chaotic regime of parameter space, we show that noise facilitates mode-hopping in all regimes. However, when the deterministic system is driven by chaotic dynamics, hops between modes are erratic and short-lived, whereas in experiments, the system spends several periods in one entrainment mode before hopping and rarely visits more than two modes. The experimental behavior matches our simulations of noise-induced mode-hopping outside the chaotic regime. We suggest that mode-hopping is a mechanism by which different NF-κB-dependent genes under frequency control can be expressed at different times.

Previous studies have suggested that changes in extracellular ion concentrations initiate the transition from an activity state that characterizes sleep in cortical neurons to states that characterize wakefulness. However, because neuronal activity and extracellular ion concentrations are interdependent, isolating their unique roles during sleep-wake transitions is not possible in vivo. Here, we extend the Averaged-Neuron model and demonstrate that, although changes in extracellular ion concentrations occur concurrently, decreasing the conductance of calcium-dependent potassium channels initiates the transition from sleep to wakefulness. We find that sleep is governed by stable, self-sustained oscillations in neuronal firing patterns, whereas the quiet awake state and active awake state are both governed by irregular oscillations and chaotic dynamics; transitions between these separable awake states are prompted by ionic changes. Although waking is indicative of a shift from stable to chaotic neuronal firing patterns, we illustrate that the properties of chaotic dynamics ensure that the transition between states is smooth and robust to noise.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.

No abstract

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

The photoautotrophic freshwater cyanobacterium Synechococcus elongatus is widely used as a chassis for biotechnological applications as well as a photosynthetic bacterial model. In this study, a method for expanding the genetic code of this cyanobacterium has been established, thereby allowing the incorporation of unnatural amino acids into proteins. This was achieved through UAG stop codon suppression, using an archaeal pyrrolysyl orthogonal translation system. We demonstrate incorporation of unnatural amino acids into green fluorescent protein with 20 ± 3.5% suppression efficiency. The introduced components were shown to be orthogonal to the host translational machinery. In addition, we observed that no significant growth impairment resulted from the integration of the system. To interpret the observations, we modeled and investigated the competition over the UAG codon between release factor 1 and pyl-tRNA. On the basis of the model results, and the fact that 39.6% of the stop codons in the S. elongatus genome are UAG stop codons, the suppression efficiency in S. elongatus is unexpectedly high. The reason for this unexpected suppression efficiency has yet to be determined.