Viral DNA packaging at base pair resolution.

摘要

During its lifecycle the bacterial virus ϕ29 packages its genome into a preformed protein shell, compressing the genome to near-crystalline density and high macroscopic pressures against large elastic, electrostatic, and entropic forces. At the core of this process is a molecular DNA pump---a complex assembly of protein and nucleic acid which extracts the chemical energy stored in the gamma phosphate bond of adenosine triphosphate and converts this energy into the mechanical work necessary to overcome these large energetic barriers.;In this thesis, we construct an optical tweezers capable of observing single base pair length changes to molecules of DNA, distance changes of only 3.4 A, and use this instrument to detect the discrete increments of DNA packaged each cycle of the packaging motor. We find that the full cycle involves the action of four of its five ATPase subunits, each of which binds ATP, delays the utilization of this molecule until the entire ring is loaded, and then packages the DNA in a 2.5-bp step, generating a collective burst of four steps each full cycle. Not only do these results indicate an intricate form of inter-subunit coordination novel for ring ATPases, they represent the first time that a molecular motor has been observed to move in a non-integer repeat of the chemical periodicity of its substrate. Both observations have profound implications for the mechanism of the packaging motor and, perhaps, related ring ATPases.;In parallel, we develop a series of new theoretical tools to extract kinetic information from the statistical properties of the inherent fluctuations in the packaging motor dynamics. In particular, we show that there are multiple classes of enzymatic fluctuations, and we provide methods for identifying the class of fluctuations in both theoretical models and actual experimental data. In parallel, we derive a Michaelis-Menten-like expression for fluctuations. We then use this expression to reveal new mechanistic properties of the packaging motor from fluctuations in the data collected. With the combination of experimental and theoretical tools developed in this thesis, the door is now open to a detailed, Angstrom-scale dissection of the mechanism of a wide variety of nucleic acid motors.