Macromolecular Crowding effects on DNA mechanics, topology and transcription - Effects of macromolecular crowding on DNA mechanics, topology, transcription, and condensation ABSTRACT Macromolecular crowding (MMC) changes the concentration and affinities of intracellular biomolecules and promotes liquid phase separation. MMC has been shown to change the melting temperature of DNA oligos, but broad characterization of how it affects the mechanical stability of DNA is incomplete. Crowded DNA condensates may generate sub-piconewton retractile tension on DNA, which can be conveniently explored using magnetic/optical tweezers. While many experiments on DNA motors employ tensions opposing or assisting translocation of several to tens of pN, our group showed that sub-piconewton tension affects DNA topology, from supercoiling to protein-mediated looping, as well as the probability that an elongating E. coli RNA polymerase (RNAP) surpasses a protein roadblock. Surprisingly, the effect of MMC on topologies such as supercoiling and protein-mediated loops, and processes such as transcription, protein spreading, and condensation has not been well characterized. This proposal aims to assess the effects of MMC on DNA configurations including unwinding and looping, protein spreading, and liquid phase separation to integrate these features into our understanding of intracellular molecular biology. To do so, we integrate single-molecule, in vitro experiments with in vivo measurements and computational/theoretical approaches Over the next five years, we will analyze both model and/or novel systems with single-molecule techniques to learn how MMC changes DNA structure, affects protein-mediated looping, and alters transcription. We will also investigate how MMC influences ParB-mediated spreading along DNA and liquid-liquid phase separation (LLPS) which requires crowding agents in vitro. Then we propose to build artificial LLPS systems with which to learn what components are required to localize a liquid-liquid phase separated droplet on a DNA segment. P-granules, Cajal bodies, segrosome, and the nucleolus are some examples of LLPS that include specific genomic regions and demonstrate the ubiquity and importance of this phenomenon. Macromolecular crowding generates forces that affect fundamental DNA mechanics and topology and in the last decade MMC has emerged as a driver of LLPS. We will integrate in vitro experiments with computational and theoretical approaches and compare with appropriate in vivo measurements performed by a collaborator. Discovering the mechanisms by which crowding modifies DNA configurations, transactions, and segregation will advance our understanding of genome biophysics and regulation and provide new tools for synthetic biology.
