Structure and NTPase activity of the RNA-translocating protein (P4) of bacteriophage φ6

Abstract

The RNA polymerase complex of bacteriophage φ6 comprises four proteins, P1, P2, P4 and P7, and forms the core of the virion. Protein P4 is a non-specific NTPase that provides the energy required for RNA translocation (packaging). Characterization of purified recombinant P4 shows that the protein assembles into stable hexamers in the presence of ADP and divalent cations. Image averaging of electron micrographs reveals this hexamer as a slightly skewed ring with outer and inner diameters of 12 and 2 nm, respectively. NTPase activity of P4 is associated only with the hexameric form. Ca 2+and Zn 2+and non-specific single-stranded RNA stimulate the NTPase activity, while Mg 2+acts as a non-competitive inhibitor, presumably via a separate Mg 2+binding site. Binding affinities of different nucleotide mono-, di- and triphosphates and non-hydrolyzable analogs indicate that the β-phosphate moiety is required for substrate binding. A slight preference for binding of purine nucleotides is also observed. Analysis of P4 by CD and Raman spectroscopy indicates an α/β subunit fold that is altered only slightly by hexamer assembly. Raman markers of P4 secondary and tertiary structures are also largely invariant to nucleotide exchange and hydrolysis, suggesting that the mechanism of RNA translocation involves movement of subunits relative to one another rather than large scale changes in the α/β subunit fold. The stoichiometry of P4 in the mature φ6 virion is estimated as 120 copies. Because the recombinant P4 hexamers exhibit hydrodynamic and enzymatic properties that are identical to those of P4 oligomers released from native φ6, we propose that P4 occurs as hexamers in the native viral core particle.