Figure 4.

Two modes of primer synthesis catalyzed by gene 4 protein. (A) Effect of 3′-flanking sequence on the primase activity of gene 4 protein. Both the 6-nt (5′-GGGTCA-3′) and 15-nt (5′-GGGTCA₁₀-3′) templates have the same primase recognition site but differ in the 3′-side flanking length. The indicated gene 4 proteins were incubated with the template in the standard reaction with or without 0.1 mM dTTP for 20 min at 37°C. The protein concentration of either the 63-kDa protein or the primase fragment was 0.1 μM, and total concentration of equimolar mixture of the 56-kDa protein and gp4-K122A was 0.3 μM. Incorporation efficiency of [α-³²P]CMP into the product is presented as height of bars in graphs. The presence or absence of dTTP is denoted. (B) Effect of defective gene 4 proteins on the primase activity of 63-kDa gene 4 protein. On the two different sets of templates described above, increasing concentrations of the 63-kDa protein alone were incubated in the standard reaction at 37°C for 20 min (▵). Either the 56-kDa protein (○) or gp4-K122A (●) was added to the 63-kDa protein to yield a total 0.3 μM protein. The mixtures of proteins used for the 15-nt template were incubated for 10 min at 37°C before the primer synthesis reaction. Incorporation of [α-³²P]CMP into the product is plotted against the 63-kDa protein concentration. (C) Effect of mixing ratio on primer synthesis catalyzed by the 56-kDa protein and g-K122A. Reaction condition was the same as the one used for the 15-mer template in B.