STRUCTURE OF VIRAL DNA PACKAGING MACHINE & PACKAGING INTERMEDIATES

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Bacteriophage 1029 of A subtilis is an excellent model for studies of viral DNA packaging. The in vitro packaging system of (029 DNA is as efficient as in vivo assembly. Models of DNA packaging can be tested and the structum of components can be determined. 1029 DNA with covalently bound gene product 3 at each terminus (DNA-gp3) is translocated in vitro into a preformed prvteir~shell (prohead) by a machine that includes the prohead'portal vertex Oead-tail comector), the 174-base 4029-encoded prohead RNA.and the ATPase gp16. The prohead, connector, pRNA and gp16 are all overproduced from cloned genes permitting the structure of these components in sequential interactions with DNA-gp3 to be studied. Supercoiled pBR322 DNA wraps around the outside of the isolated 4~29 connector. This is hypothesized to reflect the initial phase of DNA packaging. In this model, proheads should also bind supercoiled DNA and 40 DNA-gp3 should be supercoiled as a prerequisite for packaging. , An RNA pseudoknot, an intramolecular tertiary interaction, in the pRNA was inferred and then confirmed by genetic mutations. Two mutants, neither of winch can make the mtramolecular pseudoknot, can complement each other in the prohead packaging assay indicating they might make an intermolecular psuedoknot. Masses of different pRNAs were measured in the STEM to try to confirm this. It is planned to look at pRNA-connector complexes in the STEM.