FIG. 1.

Purified φ6 P2 polymerase catalyzes the transfer of an α-phosphate from a donor UTP to an acceptor ssRNA molecule. (A) Native agarose gel electrophoresis of reactions with purified P2^WT and P2^SAD assayed under standard polymerization reaction conditions in the presence of [α-³²P]UTP and a single-stranded φ6 s⁺ segment. The reactions were carried out with or without unlabeled NTPs as indicated. The positions of the labeled single-stranded (s⁺, m⁺, and l⁺) and double-stranded (S, M, and L) φ6 genome segments produced by nucleocapsid transcription (41) are shown on the left (lane N). (B) Absorbance (A₂₈₀) elution profile of P2^WT and P2^SAD separated on a Superdex 200 gel filtration column (Amersham Biosciences). The arrows indicate the P2 injection time (Inject) and the position of selected gel filtration molecular mass markers (Sigma): BD, blue dextran (2,000 kDa); Tg, thyroglobulin (669 kDa); β-Am, β-amylase (200 kDa); A, albumin (66 kDa); and CA, carbonic anhydrase (29 kDa). The lower peak corresponds to nonionic detergents of the P2 storage buffer. (Insert) Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein content in fractions 29 to 42 (upper panel, P2^WT; lower panel, P2^SAD). φ6 polymerase complex proteins (P1, P2, P4, and P7) are marked on the left. (C) Enzymatic activity in Superdex 200 fractions 25 to 44 of P2^WT (upper panel) and P2^SAD (lower panel) with s⁺₁₃₊ ssRNA template and [α-³²P]UTP (in the absence of other NTPs). (D and E) Effect of the reaction conditions on the α-³²P transfer activity of φ6 P2. The RNA products of the reaction mixtures containing sΔ⁺₁₃₊ ssRNA were separated by electrophoresis in a native agarose gel and analyzed by phosphorimager quantification. The graphs are normalized to the highest value (1) within each panel. The effect of the NH₄OAc concentration on α-³²P transfer activity is shown in panel D together with the replication activity under the same conditions for comparison. The MnCl₂ and MgCl₂ concentration effects are shown in panel E. The α-³²P transfer activity under the optimal replication reaction conditions (2 mM MnCl₂ and 5 mM MgCl₂) is indicated by a filled square.