tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control

CCA addition to the 3′ end is an essential step in tRNA maturation. High-resolution crystal structures of the CCA enzymes reveal primary enzyme contact with the tRNA minihelix domain, consisting of the acceptor stem and T stem-loop. RNA and DNA minihelices are efficient substrates for CCA addition in steady-state kinetics. However, in contrast to structural models and steady-state experiments, we show here by single-turnover kinetics that minihelices are insufficient substrates for the Escherichia coli CCA enzyme and that only the full-length tRNA is kinetically competent. Even a nick in the full-length tRNA backbone in the T loop, or as far away from the minihelix domain as in the anticodon loop, prevents efficient CCA addition. These results suggest a kinetic quality control provided by the CCA enzyme to inspect the integrity of the tRNA molecule and to discriminate against nicked or damaged species from further maturation.