Identification of bacteriophage phi 29 prohead RNA domains necessary for in vitro DNA-gp3 packaging

Functional domains of the bacteriophage phi 29 prohead RNA (pRNA) that are essential for in vitro packaging of DNA-gp3 into the prohead were mapped using pRNA mutants. Oligonucleotide-directed mutant pRNAs were produced that contained deletions and sequence alterations but were predicted to retain the overall secondary structure of wild-type pRNA. Mutant pRNAs were compared to wild-type pRNA for prohead binding in a competition assay and for DNA packaging in the defined in vitro system. The prohead binding site was previously localized to residues 22-84 on the 120-residue domain I of pRNA by ribonuclease footprinting (Reid, R. J. D., Bodley, J. W., and Anderson, D. (1994) J. Biol. Chem. 269, 5157-5162). Mutations of pRNA within the prohead binding site resulted in substantial loss of prohead binding capacity, while mutations outside of the footprint had moderate effects on prohead binding. DNA-gp3 packaging activity was correlated with pRNA binding activity for mutations within the footprint. This mutational analysis showed that both sequence and secondary structure of residues 40-91 of pRNA were crucial for prohead binding and that elements of the A helix formed from residues 1-28 and 117-92 were needed for DNA packaging functions other than prohead binding.