STRUCTURE AND NTPASE ACTIVITY OF THE RNA-TRANSLOCATING PROTEIN (P4) OF BACTERIOPHAGE-PHI-6

The RNA polymerase complex of bacteriophage phi 6 comprises four prote ins, P1, P2, P4 and P7, and forms the core of the virion. Protein P4 i s a non-specific NTPase that provides the energy required. for RNA tra nslocation (packaging). Characterization of purified recombinant P4 sh ows that the protein assembles into stable hexamers in the presence of ADP and divalent cations. Image averaging of electron micrographs rev eals this hexamer as a slightly skewed ring with outer and inner diame ters of 12 and 2 nm, respectively. NTPase activity of P4 is associated only with the hexameric form. Ca2+ and Zn2+ and non-specific single-s tranded RNA stimulate the NTPase activity, while Mg2+ acts as a non-co mpetitive inhibitor, presumably via a separate Mg2+ binding site. Bind ing affinities of different nucleotide mono-, di-and triphosphates and non-hydrolyzable analogs indicate that the beta-phosphate moiety is r equired for substrate binding. A slight preference for binding of puri ne nucleotides is also observed. Analysis of P4 by CD and Raman spectr oscopy indicates an alpha/beta subunit fold that is altered only sligh tly by hexamer assembly. Raman markers of P4 secondary and tertiary st ructures are also largely invariant to nucleotide exchange and hydroly sis, suggesting that the mechanism of RNA translocation involves movem ent of subunits relative to one another rather than large scale change s in the alpha/beta subunit fold. The stoichiometry of P4 in the matur e phi 6 virion is estimated as 120 copies. Because the recombinant P4 hexamers exhibit hydrodynamic and enzymatic properties that are identi cal to those of P4 oligomers released from native phi 6, we propose th at P4 occurs as hexamers in the native viral core particle. (C) 1998 A cademic Press Limited.