title: Translocation and rotation of tRNA during template-independent RNA polymerization by tRNA nucleotidyltransferase.
abstract: The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure. We now present crystal structures representing CC addition onto tRNA by A. aeolicus CC-adding enzyme. After C₇₄ addition in an enclosed active pocket and pyrophosphate release, the tRNA translocates and rotates relative to the enzyme, and C₇₅ addition occurs in the same active pocket as C₇₄ addition. At both the C₇₄-adding and C₇₅-adding stages, CTP is selected by Watson-Crick-like hydrogen bonds between the cytosine of CTP and conserved Asp and Arg residues in the pocket. After C₇₄C₇₅ addition and pyrophosphate release, the tRNA translocates further and drops off the enzyme, and the CC-adding enzyme terminates RNA polymerization.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Seisuke
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Daijiro
authors.initials: D
authors.lastname: Takeshita
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address: kozo-tomita@aist.go.jp.
authors.forename: Kozo
authors.initials: K
authors.lastname: Tomita
mesh_terms: D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001419:Bacteria / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D058105:Polymerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011755:Pyrophosphatases / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D012399:Rotation
chemical_list: D012313:RNA; D001216:Asparagine; D003596:Cytosine; D012343:RNA, Transfer; D001120:Arginine; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D011755:Pyrophosphatases