The role of portal and terminase proteins in the mechanism of DNA packaging in the dsDNA bacteriophage T4

Abstract

Several of the double-stranded DNA bacterial viruses have been developed into model systems for the study of basic biological and macromolecular functions. While the DNA packaging processes of bacteriophage have been well studied, the exact mechanism by which the energy derived from nucleotide hydrolysis is converted into a translocation of DNA remains unknown. In phage T4, gp20 serves as the donut-shaped, dodecameric portal protein through which DNA passes into the head, and with the gp16 and gp17 terminase proteins, forms a 'packasome' complex capable of translocating DNA. In this dissertation, two independent experimental approaches are described which investigate the role of the portal protein, gp20 and the terminase protein, gp17 in DNA packaging. To examine the function of the gp20 portal in phage T4, and to test the portal rotation model for DNA packaging, we created and incorporated gp20-fusion proteins into phage. 20-GFP (green fluorescent protein), 20-HOC (T4 highly antigenic outer capsid protein), 20-GFP-HOC, and HOC-20 fusions, when expressed in vivo, complement amber mutant defective infecting virus and produce progeny phage. Normally defective 20am mutant phage (20 am(E481) res12, 20am(N50) res325, and 20 am(B8) res492) make progeny phage in bacteria that express these portal fusions if the fusions are co-assembled with either (1) shortened, co-expressed fusion forms from the expression vector, or (2) shortened, near wild-type sized gp20 forms from infecting 20am(E481) or 20am(B8) phage. Surprisingly, fusions made to either gp20 terminus could function to make progeny phage, despite the varied roles of gp20 during phage maturation. Furthermore, N-terminally fused Hoc-20 protein complemented 20amHocam infecting phage, and analyses demonstrated the Hoc protein was positioned exteriorly and bound to its capsid binding site, essentially tethering gp20 portal in position. The ability of large protein fusions to function in making phage argues against an active, or rotating role for the gp20 portal, and suggests such a mechanism may not operate in the packaging of DNA in T4. To investigate the activities of the large subunit terminase, gp17 (70kDa), the gp17 gene was cloned, and a successful method of protein expression and purification was developed. This new purification is a significant improvement over previous methods, and yields pure, soluble gp17, active in in vitro DNA packaging assays. which show titers of 108 phage per mL of extract. (Abstract shortened by UMI.)