Project Summary/Abstract Bacterial chromosomes are organized spatially and topologically within cells, taking on conformations that change as a function of cellular processes, growth rates and conditions external to the cell. Nucleoid- associated proteins facilitate this organization by bending, looping, and coating DNA to form topological domains at different length levels. At the lowest level, the E. coli chromosome forms small looping domains, or chromosomal topologically isolated domains (TIDs), on the order of 10 kbp. This chromosomal organization into topologically isolated domains localizes supercoiling to different portions of the chromosome. The relationship between this and transcription is one of coupling, where supercoiling density determines transcriptional activity, but transcriptional activity also changes supercoiling density. To probe these effects and to test our hypothesis that the formation of TIDs significantly modulates gene expression profiles we will use a combined single-molecule imaging and computational modeling approach. To learn the most about the DNA topological effects on transcription as we must be able to isolate the various components in a series of synthetic systems. In Aim 1, I will construct an in vivo synthetic looping domain to investigate the effect of domain formation on the transcription and expression dynamics of two genes inside the domain under different conditions. Expression will be monitored via single molecule fluorescence in situ hybridization and live protein expression from synthetic looping domains. In Aim 2, to obtain a quantitative understanding of how the topological state of a TID impacts RNAP’s transcription kinetics, I will monitor the initiation and elongation rates of single RNAP molecules on a circular template DNA mimicking a TID using single molecule protein induced fluorescent enhancement (smPIFE) in vitro. In Aim 3, to synthesize the models at these two size scales by using a reaction-diffusion model to simulate DNA-protein interactions and supercoiling. Studying bacterial chromosomal organization in this way may reveal mechanisms that bacteria use to maintain transcription in the presence of perturbations and ways they may exploit DNA topology effects for gene regulation.