FIG. 4.

Cid1 complexes prefer to incorporate UMP over AMP. (A) Cid1 complexes were incubated with a 5′-labeled (A)₁₅ primer and various nucleotides, as follows: 0.5 mM ATP, 0.5 mM UTP, or 0.5 mM ATP with 0.05 mM CTP, 0.05 mM GTP, and 0.05 mM UTP (ATP, UTP, or ALL, respectively). These reaction products were then incubated with RNase A, RNase T₁, or both, as indicated, and then separated by gel electrophoresis. (B and C) Under noncompetitive conditions (Non-Comp.), Cid1 complexes were incubated with [α-³²P]ATP with 0.5 mM ATP or with [α-³²P]UTP with 0.05 mM UTP. Under competitive conditions (Comp.), Cid1 complexes were incubated with either [α-³²P]ATP or [α-³²P]UTP as well as 0.5 mM ATP, 0.05 mM CTP, 0.05 mM GTP, and 0.05 mM UTP. Three independent experiments were performed. Incorporation was measured by scintillation counting. (B) To determine the effect of nucleotide competition, incorporation under competitive conditions was normalized to noncompetitive conditions. (C) After normalization of the 10-fold difference in ATP and UTP levels, the ratio of incorporated [α-³²P]UMP to [α-³²P]AMP was obtained for both noncompetitive and competitive conditions.