Publications

• The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities.

  Yakunin A.F., Proudfoot M., Kuznetsova E., Savchenko A., Brown G., Arrowsmith C.H., Edwards A.M.

  In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity ... >> More

  In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni(2+)-dependent phosphatase activity toward pyrophosphate, canonical 5'-nucleoside tri- and diphosphates, NADP, and 2'-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2',3'-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg(2+), the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni(2+), the protein also produced some 3'-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nm) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg(2+), Mn(2+), or Ca(2+), but not Ni(2+). The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3'-CCA end of tRNA. << Less

  J. Biol. Chem. 279:36819-36827(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of the human CCA-adding enzyme: insights into template-independent polymerization.

  Augustin M.A., Reichert A.S., Betat H., Huber R., Moerl M., Steegborn C.

  All tRNA molecules carry the invariant sequence CCA at their 3'-terminus for amino acid attachment. The post-transcriptional addition of CCA is carried out by ATP(CTP):tRNA nucleotidyltransferase, also called CCase. This enzyme catalyses a unique template-independent but sequence-specific nucleoti ... >> More

  All tRNA molecules carry the invariant sequence CCA at their 3'-terminus for amino acid attachment. The post-transcriptional addition of CCA is carried out by ATP(CTP):tRNA nucleotidyltransferase, also called CCase. This enzyme catalyses a unique template-independent but sequence-specific nucleotide polymerization reaction. In order to reveal the molecular mechanism of this activity, we solved the crystal structure of human CCase by single isomorphous replacement. The structure reveals a four domain architecture with a cluster of conserved residues forming a positively charged cleft between the first two domains. Structural homology of the N-terminal CCase domain to other nucleotidyltransferases could be exploited for modeling a tRNA-substrate complex. The model places the tRNA 3'-end into the N-terminal nucleotidyltransferase site, close to a patch of conserved residues that provide the binding sites for CTP and ATP. Based on our results, we introduce a corkscrew model for CCA addition that includes a fixed active site and a traveling tRNA-binding region formed by flexible parts of the protein. << Less

  J. Mol. Biol. 328:985-994(2003) [PubMed] [EuropePMC]

• CCA addition to tRNA: implications for tRNA quality control.

  Hou Y.M.

  The CCA sequence is conserved at the 3' end of all mature tRNA molecules to function as the site of amino acid attachment. This sequence is acquired and maintained by stepwise nucleotide addition by the ubiquitous CCA enzyme, which is an unusual RNA polymerase that does not use a nucleic acid temp ... >> More

  The CCA sequence is conserved at the 3' end of all mature tRNA molecules to function as the site of amino acid attachment. This sequence is acquired and maintained by stepwise nucleotide addition by the ubiquitous CCA enzyme, which is an unusual RNA polymerase that does not use a nucleic acid template for nucleotide addition. Crystal structural work has divided CCA enzymes into two structurally distinct classes, which differ in the mechanism of template-independent nucleotide selection. Recent kinetic work of the class II E. coli CCA enzyme has demonstrated a rapid and uniform rate constant for the chemistry of nucleotide addition at each step of CCA synthesis, although the enzyme uses different determinants to control the rate of each step. Importantly, the kinetic work reveals that, at each step of CCA synthesis, E. coli CCA enzyme has an innate ability to discriminate against tRNA backbone damage. This discrimination suggests the possibility of a previously unrecognized quality control mechanism that would prevent damaged tRNA from CCA maturation and from entering the ribosome machinery of protein synthesis. This quality control is relevant to cellular stress conditions that damage tRNA backbone and predicts a role of CCA addition in stress response. << Less

  IUBMB Life 62:251-260(2010) [PubMed] [EuropePMC]