UC San Diego
Studies of viral DNA packaging motor mechanism on a single molecule level via optical tweezers
- Author(s): Ordyan, Mariam
- Advisor(s): Smith, Douglas E
- et al.
The viral DNA packaging motors are among the most powerful biological machines known, and their mechanisms have been of interest for decades. The work presented in this dissertation has been dedicated to studying these mechanisms on the example of the T4 and Lambda bacteriophages, which are well known model organisms.
Custom-built optical tweezers were utilized for real time observations of single DNA molecule packaging. A force-feedback loop allowed us to study the DNA translocation fueled by ATP hydrolysis under different conditions.
The regulation of the motors grip on the DNA by the nucleotide state was investigated through a novel technique, and the interaction of the motor subunits with the DNA was shown to depend on the state of the ATP-binding pockets. This finding brings us closer to understanding the level of coordination between the subunits during translocation. The fact that the motors grip on the DNA depends on the nucleotide state also allows to judge about the state of the ATP-binding pocket based on the packaging dynamics. A novel mechanism of the clamping of the DNA by the packaging complex to prevent dissociation was identified in the course of these experiments.
The role of the putative conserved Walker A and Walker B ATP binding motifs were examined through mutagenesis studies. An extensive study of 23 different mutants in single-molecule experiments was conducted, and it was concluded that these regions are indeed involved in ATP-binding, but we also identified residues from this region that are important for ATP-alignment, catalysis of hydrolysis, and mechanochemical coupling. In addition measurements on the mutation of the glutamate residue E179 directly downstream of Walker B were performed, and it confirmed that this residue catalyzes the hydrolysis.
A conserved Loop-Helix-Loop region previously studied in this lab, and proposed to play a regulatory role in translocation velocity was also studied. 8 different mutants were probed in the single-molecule assay, and, where packaging was detected, the effect on the translocation speed was studied.
This is, to date, the most extensive mutagenesis study preformed to investigate the roles of particular amino acids in the detailed function of a viral packaging motor..