Single Molecule Optical Trapping Measurements on Nucleosomes: Kinetics, Feedback, and High Bandwidth.

摘要

The nucleosome, a protein-nucleic acid superstructure, is the lowest level of DNA packaging in eukaryotes. The nucleosome core is composed of two copies each of histones H2A, H2B, H3, and H4. Wrapping this protein core is 147 base pairs of DNA, forming a 10 nm diameter structure. Nucleosomes not only package DNA, but act as a barrier to transcription, regulating gene expression. We measure the nucleosome's response to force with optical tweezers for three intertwined goals: To measure the kinetics dictating the nucleosome's binding and unbinding to DNA, to understand the physics of biomolecules in general, and to push the technology of optical tweezers to enable new measurements. The complete kinetics of wild-type nucleosome inner-turn unwinding and rewinding are measured, determining the free energy of DNA-histone binding, and revealing an alternate unwound state accessible at physiological forces. The kinetics of a sin mutant (H4-R45H) are also characterized, and faster unwinding and slower rewinding rates are measured. The mutant's promotion of the unwrapped or unpackaged state provides a mechanistic cause for the mutant's known increase in gene expression, linking thermodynamic stability to gene expression. An axial optical trapping calibration enables these measurements. Live jump detection is applied to single molecule optical tweezers measurements, enabling large data sets and new measurement types. Capping this work, the knowledge and techniques developed are leveraged to measure the transition path of nucleosome unwinding at megahertz bandwidth.