title: tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: Marcel
authors.initials: M
authors.lastname: Dupasquier
authors.forename: Sangbumn
authors.initials: S
authors.lastname: Kim
authors.forename: Konstantine
authors.initials: K
authors.lastname: Halkidis
authors.forename: Howard
authors.initials: H
authors.lastname: Gamper
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase