FIGURE 4.

RNase T-catalyzed tRNA end turnover. (A) Time-dependent 3′-end turnover of a 5′-end labeled tRNA^Ala transcript ending with the CCA triplet. RNase T specifically catalyzes the removal of the terminal A residues and leaves the CC sequence intact. C, control reactions incubated without enzyme for the maximum reaction time; M, size marker tRNA^Ala, 73 nt. (B) Apparent kinetics of tRNA 3′-end turnover. Increasing amounts of tRNA^Ala–CCA were incubated with RNase T under steady-state conditions and product formation was determined by quantifying the intensity of the tRNA^Ala–CC band. Michaelis–Menten parameters were determined using GraphPadPrism software (Table 2); k is the reaction rate normalized to the total enzyme concentration. Data are means ± SD; n = 3.