Single molecule force experiments investigating the role of substrate structure in a viral DNA translocase reveal a novel mechanism of translocation

Molecular machines are small assemblies harnessed to perform mechanical work in cells. These motors convert the chemical energy of ATP to move cargoes, transcribe RNA, and pump protons, to name a few examples. The study of these nanomachines is at the border of physics and biology, interesting to both fields for its implications of energy transfer at these length scales and their importance to cellular function, respectively. Optical tweezers is a natural choice as the tool to investigate these machines, as the technique has very high spatio-temporal resolution, enough to resolve the quick, small steps that these motors can take, and the applied force can be used to investigate the energetics and force generation properties of the system.The DNA packaging motor of Bacillus subtilis phage φ29 is a model system for studying biological nanomachines. The role of this homopentameric ring motor is to package the DNA genome into capsids during viral replication. This ATPase is a member of the additional strand, conserved glutamate (ASCE) superfamily, meaning the study of this motor can reveal insights to the function of other members in the family. Benefits to studying φ29 include the simplicity of performing experiments, only requiring the motor-capsid complex, DNA, and ATP, its relatively large step size (0.85nm), and moderate speed (40nm/s) which makes observation of motor stepping well-suited for optical tweezers experiments. Single-molecule optical tweezers studies of this motor have revealed great detail about its mechanochemical cycle and packaging mechanism, including interesting symmetry breaking from this homomeric ring, in which one of the five subunits is special in that it does not perform a mechanical task, but a regulatory one. This motor operates under a dwell-burst cycle, where the motor, starting from an ADP-filled ring, first exchanges ADP for ATP in an ordered, sequential fashion (during the dwell). After all five have exchanged, the special subunit hydrolyzes its ATP, which then causes the other four subunits to sequentially hydrolyze their ATPs and translocate 0.85nm (2.5bp) of substrate each (during the burst), resulting in the packaging of 10bp. The delineation between the one special and four translocatory subunits shows a division of labor, where one subunit takes on a different role from the others, despite their identical protein sequences. The similarity between the amount of DNA packaged in one cycle (10bp) and the pitch of DNA (10.4bp) is not just a coincidence, previous studies suggest that the event that assigns the identity of the special subunit is that it contacts two adjacent phosphates every pitch, and that this identity is preserved cycle after cycle. Despite this knowledge, the actual motions of the motor subunits that translate into substrate translocation are unknown. One proposed translocation model for p29 involves a dehydration-induced B-form DNA to A-form DNA transition. This scrunchworm model explains the 0.85nm step size as the difference in contour length of 10bp B-form and 10bp A-form DNA. We can test if this model is correct by making the motor package dsRNA, a substrate that can only adopt an A-form structure. In addition, this substrate has a different pitch than DNA, so we can see if the burst size changes if the pitch of the substrate changes. We found that, when challenged with dsRNA, the motor is indeed able to package it, invalidating the scrunchworm model. Packaging of dsRNA and RNA:DNA hybrid combinations reveals that the motor conforms its burst size to the periodicity of its substrate. Hence, we conclude that the motor’s burst size is determined by the pitch of the substrate. However, we observe that the step size of the motor is still the 0.85nm it is on DNA, with one step shortened to accommodate the shorter total burst size, suggesting that the step size of the motor is innate and not determined by the substrate. Because the structure of the ATP-full motor was found to exist in an open lock-washer state by cryo-EM, we propose a model of translocation in which the ring is planar at the start of the dwell and successively opens up upon nucleotide exchange during the dwell. Thus, at the end of the dwell, and at the beginning of the burst, the motor adopts a lock-washer conformation. The open-ring structure is the mechanism by which the burst size of the motor adapts to the pitch of its substrate. During the burst, nucleotide hydrolysis successively closes the ring, translocating the substrate and reverting the motor to a planar conformation. The step size is determined by the motion of the “hinges” formed by the adjacent subunits in the spiral. This translocation mechanism is an interesting example of a motor adapting to the periodicity of its substrate, while taking advantage of its repeating chemical moieties by using them as rest points like the rungs of a ladder.

Advisor: Bustamante, Carlos