(A) A schematic drawing of the tRNA75C substrate for A76 addition, using ATP as the nucleotide donor. The tRNA is labeled during transcription by incorporation of [α-32P]AMP, indicated by red dots. (B) Representative time courses of A76 addition to tRNA75C by increasing concentration of EcCCA (0.01-10 μM, as shown). Reactions were performed by pre-incubating EcCCA with tRNA75C (maintained at 1/10 below the concentration of EcCCA), followed by rapid mixing with ATP (1 mM) over various time periods, then terminated by mixing with 7 M urea as shown in (C). (C) Fitting the data of (B) to a hyperbolic equation to derive kA76 and Kd for tRNAC75.