Understanding the molecular mechanisms of transcriptional bursting.

In recent years, it has been experimentally established that transcription, a fundamental biological process that involves the synthesis of messenger RNA molecules from DNA templates, does not proceed continuously as was expected. Rather, it exhibits a distinct dynamic behavior of alternating between productive phases when RNA molecules are actively synthesized and inactive phases when there is no RNA production at all. The bimodal transcriptional dynamics is now confirmed to be present in most living systems. This phenomenon is known as transcriptional bursting and it attracts significant amounts of attention from researchers in different fields. However, despite multiple experimental and theoretical investigations, the microscopic origin and biological functions of the transcriptional bursting remain unclear. Here we discuss the recent developments in uncovering the underlying molecular mechanisms of transcriptional bursting and our current understanding of them. Our analysis presents a physicochemical view of the processes that govern transcriptional bursting in living cells.

Long-Range Supercoiling-Mediated RNA Polymerase Cooperation in Transcription.

It is widely believed that DNA supercoiling plays an important role in the regulation of transcriptional dynamics. Recent studies show that it could affect transcription not only through the buildup and relaxation of torsional strain on DNA strands but also via effective long-range supercoiling-mediated interactions between RNA polymerase (RNAP) molecules. Here, we present a theoretical study that quantitatively analyzes the effect of long-range RNAP cooperation in transcription dynamics. Our minimal chemical-kinetic model assumes that one or two RNAP molecules can simultaneously participate in the transcription, and it takes into account their binding to and dissociation from DNA. It also explicitly accounts for competition between the supercoiling buildup that reduces the RNA elongation speed and gyrase binding that rescues the RNA synthesis. The full analytical solution of the model accompanied by Monte Carlo computer simulations predicts that the system should exhibit transcriptional bursting dynamics, in agreement with experimental observations. The analysis also revealed that when there are two polymerases participating in the elongation rather than one, the transcription process becomes much more efficient since the level of stochastic noise decreases while more RNA transcripts are produced. Our theoretical investigation clarifies molecular aspects of the supercoiling-mediated RNAP cooperativity during transcription.

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils.

The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

A Mechanochemical Model of Transcriptional Bursting.

Populations of genetically identical cells generally show a large variability in cell phenotypes, which is typically associated with the stochastic nature of gene expression processes. It is widely believed that a significant source of such randomness is transcriptional bursting, which is when periods of active production of RNA molecules alternate with periods of RNA degradation. However, the molecular mechanisms of such strong fluctuations remain unclear. Recent studies suggest that DNA supercoiling, which happens during transcription, might be directly related to the bursting behavior. Stimulated by these observations, we developed a stochastic mechanochemical model of supercoiling-induced transcriptional bursting in which the RNA synthesis leads to the buildup of torsion in DNA. This slows down the RNA production until it is bound by the enzyme gyrase to DNA, which releases the stress and allows for the RNA synthesis to restart with the original rate. Using a thermodynamically consistent coupling between mechanical and chemical processes, the dynamic properties of transcription are explicitly evaluated. In addition, a first-passage method to evaluate the dynamics of transcription is developed. Theoretical analysis shows that transcriptional bursting is observed when both the supercoiling and the mechanical stress release due to gyrase are present in the system. It is also found that the overall RNA production rate is not constant and depends on the number of previously synthesized RNA molecules. A comparison with experimental data on bacteria allows us to evaluate the energetic cost of supercoiling during transcription. It is argued that the relatively weak mechanochemical coupling might allow transcription to be regulated most effectively.

Theoretical Investigation of Transcriptional Bursting: A Multistate Approach.

Variability in gene expression causes genetically identical cells to exhibit different phenotypes. One probable cause of this variability is transcriptional bursting, where the synthesis of RNA molecules randomly alternates with periods of silence in the transfer of genetic information. Yet, the molecular mechanisms behind this variability remain unclear. Experiments indicate that multiple biochemical states might be involved in the production of RNA molecules. Stimulated by these observations, we developed a theoretical framework to investigate the mechanisms of transcriptional bursting. It is based on a multistate stochastic approach that provides a full quantitative description of the dynamic properties in the system. We found that the degree of stochastic fluctuations during transcription directly correlates with the number of biochemical states. This explains experimentally observed variability and fluctuations in the quantities of the produced RNA molecules. The procedure to estimate the number of relevant biochemical states participating in the transcription is outlined and applied for analysis of experimental results. We also developed a general dynamic phase diagram for the transcription process. The presented theoretical method clarifies physical-chemical aspects of the transcriptional bursting and presents a minimal chemical-kinetic description of the process.