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 p redicted 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 bind ing site result ed in substantial loss of prohead binding capacity, while mutations ou tside of the footprint had moderate effects on prohead binding. DNA gp 3 packaging activity was correlated with pRNA binding activity for mut ations within the footprint. This mutational analysis showed that both sequence and secondary structure of residues 40-91 of pRNA were cruci al for prohead binding and that elements of the A helix formed from re sidues 1-28 and 117-92 were needed for DNA packaging functions other t han prohead binding.