title: Pyrophosphorolysis of CCA addition: implication for fidelity.
abstract: In nucleic acid polymerization reaction, pyrophosphorolysis is the reversal of nucleotide addition, in which the terminal nucleotide is excised in the presence of inorganic pyrophosphate (PPi). The CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3' end without using a nucleic acid template. To better understand the reaction mechanism of CCA addition, we tested pyrophosphorolysis of CCA enzymes, which are divided into two structurally distinct classes. Here, we show that only class II CCA enzymes catalyze pyrophosphorolysis and that the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Pyrophosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions. Importantly, pyrophosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3' end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. Measurement of kinetic parameters of the class II Escherichia coli CCA enzyme reveals that the enzyme catalyzes pyrophosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to PPi relative to NTP, suggesting a mechanism in which PPi is rapidly released after each nucleotide addition as a driving force to promote the forward synthesis of CCA.
authors: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors: Takao
authors: T
authors: Igarashi
authors: Cuiping
authors: C
authors: Liu
authors: Hironobu
authors: H
authors: Morinaga
authors: Sangbumn
authors: S
authors: Kim
authors: Ya-Ming
authors: YM
authors: Hou
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D003570:Cytidine Triphosphate / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase