Browsing by Author "Klindziuk, Alena"
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Item Long-Range Supercoiling-Mediated RNA Polymerase Cooperation in Transcription(American Chemical Society, 2021) Klindziuk, Alena; Kolomeisky, Anatoly B.; Center for Theoretical Biological PhysicsIt 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.Item Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils(National Academy of Sciences, 2021) Yang, David S.; Saeedi, Arash; Davtyan, Aram; Fathi, Mohsen; Sherman, Michael B.; Safari, Mohammad S.; Klindziuk, Alena; Barton, Michelle C.; Varadarajan, Navin; Kolomeisky, Anatoly B.; Vekilov, Peter G.; Center for Theoretical Biological PhysicsThe 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.Item Stochastic Modeling of DNA Transcription and Gene Expression(2021-08-09) Klindziuk, Alena; Kolomeisky, Anatoly B.Transcription is a fundamental biological process of copying genetic information from DNA to messenger RNA and is the first step in the gene expression process. Transcription is governed by a complex system of biochemical and biophysical processes and, despite decades of research, open questions about the molecular mechanisms behind transcription remain. For example, our understanding of the molecular mechanisms behind the transcriptional bursting phenomenon, an observation that messenger RNA molecules are produced in a discontinuous manner, and our understanding of gene expression pattern formation during embryonic development is still incomplete. The chapters of this thesis are dedicated to developing various mechanistic and phenomenological models of transcription. Since transcription typically involves small numbers of products and random interactions between the reactants, we adopt a stochastic, chemical kinetic approach for our investigations. First, following an observation that transcription involves a spectrum of activity states, we develop a phenomenological, multi-state model of transcription. Using this model, we draw a connection between the number of independent biochemical states of transcription and the number of maxima in the distribution of transcripts produced. We also develop a method of evaluating the number of transcriptional states from experimental data. Second, we develop a mechanistic model that shows that transcriptional bursting can result from the interplay between DNA supercoiling buildup and supercoiling release due to the enzymatic action of gyrase. Using the mean first-passage time analysis and experimental measurements of transcription elongation rate, we calculate the energy needed to produce an additional transcript under the supercoiling conditions. Third, following a set of observations that suggest that collective dynamics of messenger RNA-producing RNA polymerases (RNAPs) play an important role in ensuring transcriptional integrity, we develop two different mechanistic models of transcription which take into account the long-range cooperative interactions between RNAPs and show that such cooperativity can result in a simultaneous increase in transcriptional productivity and decrease in transcriptional noise at an optimal value of mechanochemical coupling. Lastly, in response to recent observations that spatial modulation in chemical kinetic rates of transcript production and promoter activation is associated with gene expression pattern formation in embryos, we developed a phenomenological model that demonstrates that transcriptional two-state ON/OFF model with exponentially varying chemical kinetic rates of either transcript production and promoter turn ON or transcript production and promoter turn OFF rates can produce a spatial gene expression stripe pattern.Item Theoretical Investigations of Transcriptional Bursting(2020-04-22) Klindziuk, Alena; Kolomeisky, Anatoly BTranscriptional bursting, or the random alternation between periods of synthesis of messenger RNA molecules and cessation of the transfer of genetic information, is present in all cell types and is an important contributing factor to gene expression noise. Yet, its exact molecular mechanisms remain unclear. In response to an experimental observation that certain transcription systems have a spectrum of activity levels, we developed a phenomenological multi-state theoretical model of transcription. By solving the model exactly and analyzing the results, we found that the degree of stochastic fluctuations during transcription directly correlates with the number of independent biochemical states in the system. In response to studies that show that supercoiling plays an important role in bacterial transcription, we developed a more microscopic mechano-chemical model of transcription. Solving the model, we demonstrate that the interplay between chemical process of RNA synthesis and mechanical stress build up on the DNA strand can lead to transcriptional bursting dynamics. Also, the first-passage analysis was used to estimate the energy to produce an RNA when supercoiling is at play.Item Understanding the molecular mechanisms of transcriptional bursting(Royal Society of Chemistry, 2021) Klindziuk, Alena; Kolomeisky, Anatoly B.; Center for Theoretical Biological PhysicsIn 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.