tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control |
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Authors: | Dupasquier Marcel Kim Sangbumn Halkidis Konstantine Gamper Howard Hou Ya-Ming |
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Affiliation: | Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA |
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Abstract: | 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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Keywords: | AfCCA, Archaeoglobus fulgidus CCA enzyme ASL, anticodon stem-loop EcCCA, Escherichia coli CCA enzyme |
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