Characterization of the prohead-pRNA interaction of bacteriophage phi 29

The small prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi 29 is essential for ATP-dependent packaging of viral DNA. The 174-, 124-, and 120-residue forms of pRNA produced in vitro using T7 RNA polymerase were equivalent in prohead binding and DNA packaging activity to pRNAs produced in phi 29-infected cells. pRNA binding to proheads, characterized by the use of Northern hybridization and filter binding assays, was specific, rapid, and irreversible in the presence of 10 mM Mg2+. Proheads produced in phage-infected cells carried 5.8 +/- 2.7 copies of pRNA, and proheads assembled in Escherichia coli in the absence of pRNA bound 6.0 +/- 3.5 copies of pRNA. Footprints of proheads on pRNA generated with the ribonucleases A, T1, and V1 showed that nucleotides 22-84, 5' to 3', were protected from ribonuclease attack. Enhanced cleavage at nucleotides 37-40 with ribonuclease V1 suggested a conformational change of pRNA upon prohead binding.