Probing the structure of bacteriophage phi 29 prohead RNA with specific mutations

Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA genome into a preformed prohead in an ATP-dependent reaction. A 174-residue phi 29-encoded RNA molecule (pRNA) is a structural component of the prohead and is essential for DNA packaging. The secondary and tertiary structures of the prohead binding site on pRNA have been probed using a series of specific mutant pRNAs and by measuring binding to RNA-free proheads and in vitro packaging of the DNA-gene product 3 (DNA.gp3) complex. A pseudoknot in pRNA inferred from phylogenetic studies was confirmed with specific mutations, and this pseudoknot was necessary for DNA.gp3 packaging activity. pRNA was truncated progressively from the 5' and 3' ends to isolate the prohead binding site, and three truncated pRNAs of 79, 71, and 62 residues retained prohead binding activity but could not reconstitute proheads for DNA.gp3 packaging. Mutation resulting in changes of the D hairpin loop and its connecting residues within the prohead binding site of pRNA and DNA packaging studies demonstrated that some alteration of secondary structure in this helix was permissible. The analyses provided further confirmation of a discrete prohead binding domain in pRNA and further delineated specific structural requirements for DNA.gp3 packaging activity which may not be required for prohead binding.