Part I: Super-resolution Microscopy Method Development Part II: Investigations of Transcription Regulation by Chromosomal Organization in Bacteria

Part I: SMLM provides not only high-resolution images of molecular assemblies beyond the diffraction limit but also enables quantitative analysis of the dynamics and compositions. However, challenges in imaging and analysis due to cell geometry, resolution limit, and fluorophore properties impede the full potential of SMLM. To address these challenges, I first developed a single- molecule tracking methodology that minimizes the confinement of diffusing molecules to obtain accurate diffusion coefficients and transition rates. Next, I developed a methodology to improve three-dimensional (3D)-SMLM imaging by directly taking into account the variability of 3D point-spread-functions, which produces superior resolution compared to existing methodologies. Finally, I developed a method to correct for blinking-artifacts. Blinking-artifacts are caused by repeated localizations of the same fluorophores, which distort images and produce false nanoclusters. I derived a method to find the ”ground- truth” of the underlying pairwise distribution without any additional calibration. This ground truth enables me to identify the true underlying spatial distribution of molecules in the SMLM image, solving a problem that has long persisted in the field. Part II: It is well established that chromosomal organization dramatically influences transcription, but the underlying mechanisms remain elusive. We hypothesize that supercoiling constrained by the chromosomal topology has an effect on transcription rate and hence coordinates expression within the same topological domain. To examine this hypothesis, I developed a theoretical model to account directly for the buildup of supercoiling due to transcription in a DNA-loop. To investigate how the topology of the chromosome influences transcription further, I then developed the first in vivo assays to manipulate the formation of a “large” chromosomal DNA topological domain in E. coli cells to examine transcription activity of multiple genes enclosed in the domain. My experiments showed that domain formation decreases expression levels of genes both inside and outside the domain — demonstrating a ”long-range” cis-regulatory mechanism due to the “architecture” of the chromosome within bacteria. Finally, using quantitative SMLM, we investigated how ”large-scale” chromosome organization affects the spatial organization of RNA-polymerase (RNAP). We discovered RNAP clusters engaged in active ribosomal RNA synthesis; whose organization is “driven” by the chromosomal organization