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.
Reid
R J
RJ
Department of Genetics and Cell Biology, University of Minnesota, Minneapolis 55455.
Bodley
J W
JW
Anderson
D
D
D017103:Bacillus Phages / Q000254:growth & development*; D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012367:RNA, Viral / Q000502:physiology*; D013329:Structure-Activity Relationship; D014779:Virus Replication
D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral