Protein-Mediated Molecular Bridging: A Key Mechanism in Biopolymer Organization

Protein-mediated bridging is ubiquitous and essential for shaping cellular structures in all organisms. Here we dissect this mechanism for a model system: the Histone-like Nucleoid-Structuring protein (H-NS). We present data from two complementary single-molecule assays that probe the H-NS-DNA interaction: a dynamic optical-trap-driven unzipping assay and an equilibrium H-NS-mediated DNA looping scanning force microscopy imaging assay. To quantitatively analyze and compare these assays, we employ what we consider 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. We expect that this theoretical framework for describing protein-mediated bridging will be applicable to proteins acting in chromatin and cytoskeletal organization

Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA

Architectural proteins have an important role in shaping the genome and act as global regulators of gene expression. How these proteins jointly modulate genome plasticity is largely unknown. In archaea, one of the most abundant proteins, Alba, is considered to have a key role in organizing the genome. Here we characterize the multimodal architectural properties and interplay of the Alba1 and Alba2 proteins using single-molecule imaging and manipulation techniques. We demonstrate that the two paralogues can bridge and rigidify DNA and that the interplay between the two proteins influences the balance between these effects. Our data yield a structural model that explains the multimodal behaviour of Alba proteins and its impact on genome folding

H-NS promotes looped domain formation in the bacterial chromosome

SummaryThe bacterial chromosome is organized into loops, which constitute topologically isolated domains. It is unclear which proteins are responsible for the formation of the topological barriers between domains. The abundant DNA-binding histone-like nucleoid structuring protein (H-NS) is a key player in the organization and compaction of bacterial chromosomes [1,2]. The protein acts by bridging DNA duplexes [3], thus allowing for the formation of DNA loops. Here, genome-wide studies of H-NS binding suggest that this protein is directly involved in the formation or maintenance of topological domain barriers

Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation

Both prokaryotic and eukaryotic organisms contain DNA bridging proteins, which can have regulatory or architectural functions. The molecular and mechanical details of such proteins are hard to obtain, in particular if they involve non-specific interactions. The bacterial nucleoid consists of hundreds of DNA loops, shaped in part by non-specific DNA bridging proteins such as histone-like nucleoid structuring protein (H-NS), leucine-responsive regulatory protein (Lrp) and SMC (structural maintenance of chromosomes) proteins. We have developed an optical tweezers instrument that can independently handle two DNA molecules, which allows the systematic investigation of protein-mediated DNA-DNA interactions. Here we use this technique to investigate the abundant non-specific nucleoid-associated protein H-NS, and show that H-NS is dynamically organized between two DNA molecules in register with their helical pitch. Our optical tweezers also allow us to carry out dynamic force spectroscopy on non-specific DNA binding proteins and thereby to determine an energy landscape for the H-NS-DNA interaction. Our results explain how the bacterial nucleoid can be effectively compacted and organized, but be dynamic in nature and accessible to DNA-tracking motor enzymes. Finally, our experimental approach is widely applicable to other DNA bridging proteins, as well as to complex DNA interactions involving multiple DNA molecules. ©2006 Nature Publishing Group

Visualizing single DNA-bound proteins using DNA as a scanning probe

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions