In the last quarter century, biologists have made great strides towards understanding biology at the molecular scale. The human genome has been sequenced and the structures of many proteins, the molecular machines responsible for the function and structure of cells, have been solved. Single-molecule techniques and advances in microscopy have significantly changed the way in which biologists ask and answer questions. As biological measurements and techniques have become increasingly quantitative, they have allowed biologists to ask ever more quantitative questions: How do the molecular machines, which comprise the cell, function microscopically? Can we understand the design principles that govern the structure and function of biological systems on a microscopic scale? What role does the chaotic microscopic environment play in cellular function? One outcome of this new generation of quantitative biological questions is the need to greet quantitative experiments with models at a higher level of abstraction than the traditional cartoons of molecular biology. Our work centers on the interface between mathematical models of biological systems and this new generation of quantitative biological experiments.

To probe chromosome structure in vivo we built a collection of Ecoli strains that carry three spectrally-distinct fluorescently-labled genetic loci. The measurements of the locus distributions reveal that the Ecoli chromosome is precisely organized into a nucleoid filament, despite the long-standing assumption that the chromosome is well described by a polymer model. Loci in the body of the nucleoid show a precision of positioning within the cell of better than 10% the cell length. The precision of inter-locus positioning of genomically proximate loci was greater than just 4% of the cell length. The measured dependence of the precision of inter-locus position on genomic distance singles out intra-nucleoid interactions as the mechanism responsible for chromosome organization. From the magnitude of the precision of positioning, we infer the existence of an as-yet uncharacterized higher-order DNA organization in prokaryotic cells. We demonstrate that both the stochastic and average structure of the nucleoid is captured by a fluctuating elastic filament model.

H-NS: Molecular bridging by flexible linkers. The bacterial nucleoid is structured by a large number of histone-like proteins called Nucleoid Associated Proteins (NAPs). NAPs share no structural homology with histones but play a dual regulatory and structural role in the prokaryotic cell. The two dominant modes of NAP function are DNA bending (HU, IHF) and bridging (H-NS). In collaboration with the Wuite Lab, we investigated the mechanism of H-NS-induced DNA bridging using two complementary single molecule assays: a dynamic optical-trap-driven unzipping assay and an equilibrium H-NS-mediated DNA-looping assay. To quantitatively analyze and compare these assays, we employ a novel theoretical framework that describes the bridging motif. The interplay between the experiments and our theoretical model not only infers the effective interaction free energy, the bridging conformation and the duplex-duplex spacing, but also reveals a second, unresolved, cis-binding mode that challenges our current understanding of the role of bridging proteins in chromatin structure. The cis-binding mode is expected to dramatically speed up the rate of nucleoid remodeling relative to proteins that bind in trans only. We expect that the theoretical framework for describing protein-mediated bridging will be applicable to proteins acting in chromatin and cytoskeletal organization.