Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

SignificanceSeveral active processes operate on eukaryotic genomes, dictating their three-dimensional arrangement and dynamical properties. The combination of structural organization and dynamics is essential to the proper functioning of the cell. We show that an effective energy landscape model for chromatin provides a unifying description of both the structural and dynamical aspects of the genome, recapitulating many of its features. Using this quasi-equilibrium energy landscape model, we demonstrate that the physical interactions accounting for genome architecture also lead to the nontrivial dynamical behavior of genomes previously described in multiple experimental observations.

Histone tails are highly flexible N- or C-terminal protrusions of histone proteins which facilitate the compaction of DNA into dense superstructures known as chromatin. On a molecular scale histone tails are polyelectrolytes with high degree of conformational disorder which allows them to function as biomolecular "switches", regulating various genetic processes. Unfortunately, their intrinsically disordered nature creates obstacles for comprehensive experimental investigation of both the structural and dynamical aspects of histone tails, because of which their conformational behaviors are still not well understood. In this work we have carried out ∼3 microsecond long all atom replica exchange molecular dynamics (REMD) simulations for each of four histone tails, H4, H3, H2B, and H2A, and probed their intrinsic conformational preferences. Our subsequent free energy landscape analysis demonstrated that most tails are not fully disordered, but show distinct conformational organization, containing specific flickering secondary structural elements. In particular, H4 forms β-hairpins, H3 and H2B adopt α-helical elements, while H2A is fully disordered. We rationalized observed patterns of conformational dynamics of various histone tails using ideas from physics of polyelectrolytes and disordered systems. We also discovered an intriguing re-entrant contraction-expansion of the tails upon heating, which is caused by subtle interplay between ionic screening and chain entropy.

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

Liquid−liquid phase separation of multivalent intrinsically disordered protein−RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA−protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA−protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA−protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein−RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Genetic switches based on the NF-κB/ IκB/ DNA system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that IκB can actively remove NF-κB bound to its genetic sites via a process called "molecular stripping." This allows the NF-κB/ IκB/ DNA switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse-grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor NF-κB and its various binary and ternary complexes with DNA and the inhibitor IκB. These studies show that the function of the NF-κB/ IκB/ DNA genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of IκB that occurs through conformational selection. Free energy calculations for DNA binding show that the binding of IκB not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the free energy onto various reaction coordinates reveal the structural details of the stripping pathways. T he binding and release of protein transcription factors from DNA are fundamental molecular processes by which genes are regulated in the cell. The pioneering studies of Jacob, Monod, Ptashne, and Gilbert explained how these two processes, seeming inverses of each other, while being maintained in local chemical equilibrium, could still lead to robust genetic switches by coupling to protein synthesis and degradation, which are kinetically controlled far from equilibrium processes (1-4). This classic picture, with the law of mass action at its core (5, 6), suggests that understanding the molecular mechanism of the binding and release of transcription factors is of secondary interest compared with understanding the thermodynamics of protein-DNA recognition. The recent discovery of proteininduced release of a DNA-bound transcription factor in the NF-κB=IκB=DNA genetic switch changes this picture (7). The induced process, called "molecular stripping," opens up the possibility of molecular kinetic control of binding and release, thus overturning the classic paradigm based only on thermodynamic control. In this paper, we use molecular dynamics simulations of coarse-grained but predictive energy landscape models of the proteins along with their interacting DNA to explore first how the NF-κB transcription factor binds individually both to DNA and to its inhibitor IκB and then to study how an approaching IκB can strip the NF-κB from a DNA molecule to which it has already been bound, by forming an intermediate ternary complex. These simulations show that each of the binary binding events involves conformational selection of different NF-κB global conformations. Molecular stripping then occurs that is induced by forming the ternar...

Intrinsically disordered proteins (IDP) are a broad class of proteins with relatively flat energy landscapes showing a high level of functional promiscuity, which are frequently regulated through posttranslational covalent modifications. Histone tails, which are the terminal segments of the histone proteins, are prominent IDPs that are implicated in a variety of signaling processes, which control chromatin organization and dynamics. Although a large body of work has been done on elucidating the roles of posttranslational modifications in functional regulation of IDPs, molecular mechanisms behind the observed behaviors are not fully understood. Using extensive atomistic molecular dynamics simulations, we found in this work that H4 tail mono-acetylation at LYS-16, which is a key covalent modification, induces a significant reorganization of the tail's conformational landscape, inducing partial ordering and enhancing the propensity for alpha-helical segments. Furthermore, our calculations of the potentials of mean force between the H4 tail and a DNA fragment indicate that contrary to the expectations based on simple electrostatic reasoning, the Lys-16 monoacetylated H4 tail binds to DNA stronger than the unacetylated protein. Based on these results, we propose a molecular mechanism for the way Lys-16 acetylation might lead to experimentally observed disruption of compact chromatin fibers.histone tails | atomistic simulations | polyelectrolytes

The growing interest in the DNA-based mesoscale systems of biological and nonbiological nature has encouraged the computational molecular science community to develop coarse-grained (CG) representationsof the DNA that will be simple enough to permit exhaustive simulations in a reasonable amount of time, yet complex enough to capture the essential physics at play. In the recent years, there have been some major developments in the DNA coarse-graining area and several fairly sophisticated models are now available that faithfully reproduce key mechanical and chemical properties of the double-and single-stranded DNA. However, there are still many challenges, which limit the applicability of the present models, and much has to be done yet to develop more reliable schemes which would have a predictive power beyond the target domain of the intrinsic parametrization. A development of robust, controllable, and transferrable CG DNA force fields will provide an invaluable tool for gaining physical insights into the molecular nature of complex DNA-based nanoscale entities such as the chromatin, virus capsids, and DNA nanocomposites. In the present contribution, we provide an overview of the recent developments in the DNA coarse-graining field. Our aim is to review the existing CG models of the double-stranded DNA, where a small selection of models, which we believe provide avenues for promising future development, are discussed in some detail. C 2012 John Wiley & Sons, Ltd. tour length of DNA in a micrometer-sized nucleus of a eukaryotic cell is of the order of 1 m. 14 The physical basis and the biological implications of the nearly million-fold compression of the highly charged and relatively stiff macromolecule are currently actively investigated. In vivo, DNA is packed into chromatin fibers in complexation with positively charged proteins, called histones. Despite structural condensation and packing, the chromatin organization allows retrieval of the desired portions of DNA in a timely manner for processing and manipulation. 11,15 Developing a more complete physical picture of DNA in isolation as well when complexed with various proteins is needed for making further progress in uncovering the principles of genetic regulation in biology.Recently, a great interest has also emerged in the nonbiological applications of the DNA, namely, the design of the DNA-based nanoscale materials. 16 Through the pioneering works of Seeman and coworkers on DNA nanoassembly, the programmable design of the DNA nanomaterials