FIGURE 6.

Oligoribonucleotide synthesis by primase fragments with alanine replacing tryptophan. A, the standard reaction for primer synthesis contained a 0.2 μm concentration of the indicated primase fragment, 4 μm template 1 containing 5′-TGGTC-3′ (Table 1), 1 mm ATP, and 1 mm [α-³²P]CTP (0.1 mCi/ml) in buffer B. Wild-type primase fragment and primase fragments containing substitution of tyrosine or alanine for tryptophan at position 97 or 147 were used. After incubation at 23 °C for 30 min, the di-, tri-, and tetraribonucleotide products (indicated to the left) were separated, and the amount of pppACCA was measured. The percentage of activity of each protein was calculated by assigning a value of 100% for the wild-type primase fragment. B, template-independent diribonucleotide synthesis. The standard reaction contained a 2 μm concentration of the indicated primase and 1 mm [α-³²P]CTP (0.1 mCi/ml) in buffer C. After incubation at 23 °C for 1 h, the product pppCC was dephosphorylated to yield CC. The product CC was separated, and the amount was determined. The percentage of activity of each protein was calculated as described in A. C, template-directed diribonucleotide synthesis. Reactions contained 0.4 μm indicated primase, 4 μm template 3 containing 5′-AAGTC-3′ (Table 1), 1 mm ATP, and 1 mm [α-³²P]CTP (0.1 mCi/ml) in the same buffer B. After incubation at 23 °C for 30 min, the product pppAC was dephosphorylated to AC as described in B. The amount of AC synthesized by each primase fragment was determined as in B. D, template-dependent extension of diribonucleotide. The reaction contained 0.4 μm indicated primase, 4 μm template 4 containing 5′-GGGTC-3′ (Table 1), 1 mm 5′-AC-3′, and 1 mm [α-³²P]CTP (0.1 mCi/ml) in the same buffer B. After incubation at 23 °C for 30 min, the products were separated, and the amount of pppACCC was measured as in A. Representative data from multiple experiments are presented in A–D.