Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis for nucleotide selection

The 3′-terminal C analogs block addition of ATP, but not CTP analogs. (A) Addition of CTP analogs zebularine, pseudoisocytidine, and 6-azacytidine (200 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class I S. shibatae was used in this assay. (B) Addition of CTP analogs 5-fluorocytidine and 5-methylcytidine (500 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class II B. stearothermophilus and E. coli enzymes were assayed at 55°C or 37°C, respectively. (C) Addition of the CTP analog 5-fluorocytidine (200 μM) in the presence of different concentrations of [α-³²P]ATP to unlabeled tRNA substrates lacking CCA. The class II B. Stearothermophilus enzyme was assayed for 15 min at 55°C and products were resolved by 12% denaturing PAGE. (F) 5-fluorocytidine. Assays as in Figures 2 and 3.