Fig. 3.

Role of R56 and R58 in Gp2 function. (A) An autoradiograph of a 20% (wt/vol) denaturing gel showing synthesis of the transcript ApApUpU (indicated by the arrow; with the underlined nucleotides ³²P-labeled) from lacUV5 by Eσ⁷⁰ in the presence of increasing amounts of Gp2^R56A and Gp2^R58A. The percentage ApApUpU synthesized (%A) by Eσ⁷⁰ in the presence of Gp2 with respect to reactions with no Gp2 are given at the bottom of the gels. (B) An autoradiograph of a 4.5% (wt/vol) native gel showing the binding of ³²P-Gp2^WT (Top), ³²P-Gp2^R56A (Middle), and ³²P-Gp2^R58A (Bottom) to Eσ⁷⁰ are shown. The migration positions of ³²P-Gp2 (lane 1) and the Eσ⁷⁰-Gp2 complex (lanes 2–6) are indicated. Radioactivity in the mutant and WT Eσ⁷⁰-Gp2 complexes was measured, and the Eσ⁷⁰-binding activity of the R56A and R58A Gp2 mutants is expressed as the percentage of Gp2^WT-binding activity (%C) for each corresponding ratio of Eσ⁷⁰:³²P-Gp2. At the bottom of the middle and bottom panels, the percentage of mutant ³²P-Gp2 associated with Eσ⁷⁰ compared with Gp2^WT is given (%C). In A and B, the molar ratio of Gp2 present with respect to Eσ⁷⁰ in each lane is shown at the top. (C) Plating efficiency of T72 AM64 phage on E. coli strain BL21 transformed with pSW33gp2 encoding mutant Gp2 proteins with the R to E and R to K substitutions at positions 56 and/or 58.