Rjd. Reid et al., PROBING THE STRUCTURE OF BACTERIOPHAGE-PHI-29 PROHEAD RNA WITH SPECIFIC MUTATIONS, The Journal of biological chemistry, 269(28), 1994, pp. 18656-18661
Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded
DNA genome into a preformed prohead in an ATP-dependent reaction. A 1
74-residue phi 29-encoded RNA molecule (pRNA) is a structural componen
t of the prohead and is essential for DNA packaging. The secondary and
tertiary structures of the prohead binding site on pRNA have been pro
bed using a series of specific mutant pRNAs and by measuring binding t
o RNA-free proheads and in vitro packaging of the DNA-gene product 3 (
DNA.gp3) complex. A pseudoknot in pRNA inferred from phylogenetic stud
ies was confirmed with specific mutations, and this pseudoknot was nec
essary for DNA.gp3 packaging activity. pRNA was truncated progressivel
y from the 5' and 3' ends to isolate the prohead binding site, and thr
ee truncated pRNAs of 79, 71, and 62 residues retained prohead binding
activity but could not reconstitute proheads for DNA.gp3 pack aging.
Mutation resulting in changes of the D hairpin loop and its connecting
residues within the prohead binding site of pRNA and DNA packaging st
udies demonstrated that some alteration of secondary structure in this
helix was permissible. The analyses provided fur ther confirmation of
a discrete prohead binding domain in pRNA and further delineated spec
ific structural requirements for DNA.gp3 packaging activity which may
not be required for prohead binding.