| dc.description.abstract | Chromosome organization is integral to life, as it plays a pivotal role in maintaining the integrity and functionality of genomic materials, as well as facilitating the transcription, replication, and transmission of genetic information during cell division. Despite lacking membrane-based compartmentalization strategies, bacteria compactly organize their chromosomes within the nucleoid, an unpartitioned subcellular space, in a hierarchical manner. Due to this open-plan layout, macromolecular crowding (MMC) is an essential factor in bacterial chromosome organization. Indeed, it has been known that chain molecules in a crowded medium can undergo phase-separation, transitioning into collapsed states. However, the extent to which bacteria rely on MMC for organizing their chromosomes is not fully understood. Moreover, chromosomes exhibit structural heterogeneity, being decorated with various proteins such as the cross-linking protein histone-like nucleoid-structuring (H-NS) and the key transcription enzyme RNA polymerase (RNAP). The resulting chain heterogeneity can influence chromosome organization in a crowded medium, with MMC potentially modulating the effects of these proteins on their target chromosomal segments. A comprehensive understanding of chromosome organization would necessitate acquiring a fuller picture of how chain heterogeneity, MMC, and the action of DNA binding proteins (DBPs) are orchestrated in a confined space.
In this thesis, using molecular dynamics (MD) simulations, we study how biomolecular crowding, confinement, chain heterogeneity, and chain cross-linking affect the spatial organization of “chromosome-like” polymers. Our modeling efforts are inspired by the way Escherichia coli (E. coli) chromosomes are organized. For this, we start with a simple model and gradually improve our modeling strategy by incorporating more biological details. These efforts yield quantitative insights into some key observations such as the clustering of transcription-active units into a transcription factory as well as H-NS and crowder synergy in condensing bacterial chromosomes.
First, our homogeneous-polymer model establishes a relationship between its spatial organization and the distribution of the surrounding crowders under anisotropic (cylindrical) confinement. This effort extends the applicability of the previous findings for unconfined spaces to cell-like confined spaces: in a parameter space of biological relevance, the sum of the volume fractions of monomers and crowders, rescaled by their respective size, remains constant.
We then introduce chain heterogeneity, simulating the effects of transcription on an otherwise homogeneous polymer. The resulting polymer contains large monomers dispersed along the backbone with small ones in between. This effort demonstrates that the compaction transition by crowders is well correlated with the clustering: when the large monomers are of sufficient size, chain compaction and clustering of large monomers occur concomitantly at the same narrow (biologically-relevant) range of the crowder volume fraction. It also indicates that cylindrical confinement makes MMC effects more effective.
Finally, we study the action of H-NS protein and its impact on chromosome compaction. H-NS, modeled as a mobile “binder,” can bind to a chromosome-like polymer. This effort elucidates how MMC and H-NS binding each play a part in compacting a bacterial chromosome, providing a quantitative understanding of the synergistic interactions between crowders and binders. Crowders intensify the binding of H-NS to the polymer, and conversely, the presence of H-NS improves the efficiency of MMC effects, indicating a bidirectional synergy in chain compaction. Additionally, we observe that the presence of crowders facilitates the clustering of binders, where the cluster size grows as the volume fraction of crowders increases.
This thesis outlines a physical framework where phase separation and clustering, driven by MMC, are identified as the principal mechanisms in bacterial chromosome organization. | en |
