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J Mol Biol. 2008 Jun 6;379(3):579-88. doi: 10.1016/j.jmb.2008.04.005. Epub 2008 Apr 8.

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

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Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.


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.

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