Mechanism of ATP-Driven DNA Packaging in Bacteriophage T4

Catholic University Of America, Washington DC

Investigators

Abstract

The mechanism by which double stranded DNA viruses translocate DNA into a preformed virus coat and organize it into a structure that has a density as high as a DNA crystal is a fascinating, unsolved, biological problem. In bacteriophage T4, an E. coli lytic virus, a 56 mm long DNA molecule is packaged within an icosahedral capsid that is approximately 0.115 mm long and 0.085 mm wide. Genetic and biochemical studies suggest that an ATP-powered DNA packaging "machine" constituted by the prohead portal protein and two nonstructural packaging/terminase proteins drives DNA packaging. This project addresses one of the central questions of the DNA packaging problem, i.e., which component of the packaging machine consumes the ATP and couples it to DNA translocation? It is hypothesized that the large terminase subunit gp17, which possesses two consensus ATP binding sites and exhibits an ATPase activity, is the translocating ATPase in phage T4. This hypothesis will be tested by a novel combinatorial mutagenesis paradigm. Mutant libraries consisting of all possible codon combinations will be constructed at the signature residues of the consensus ATP binding sites in gp17. The mutations will be transferred into phage T4 genome by a site-specific marker rescue strategy. A collection of novel mutants exhibiting null, plaque-size, and temperature sensitive phenotypes will be generated. Selected mutants will be analyzed by molecular and biochemical approaches to test the linkage between the putative ATPase centers and DNA translocation. The predictions of a rotatory model for DNA translocation will also be tested. These approaches are broadly applicable to analyze functional sites in other terminases and ATP-triggered biochemical machines, and serve as an exciting model for training undergraduate and graduate students.