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-, 1 24-, and 120-residue forms of pRNA produced in vitro using T7 RNA poly merase were equivalent in prohead binding and DNA packaging activity t o pRNAs produced in phi 29-infected cells. pRNA binding to proheads, c haracterized by the use of Northern hybridization and filter binding a ssays, was specific, rapid, and irreversible in the presence of 10 mM Mg2+. Proheads produced in phage-infected cells carried 5.8 +/- 2.7 co pies of pRNA, and proheads assembled in Escherichia coil in the absenc e 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 nucleo tides 22-84, 5' to 3', were protected from ribonuclease attack. Enhanc ed cleavage at nucleotides 37-40 with ribonuclease V1 suggested a conf ormational change of pRNA upon prohead binding.