title: The CCA-adding enzyme has a single active site.
abstract: The CCA-adding enzyme (tRNA nucleotidyltransferase) synthesizes and repairs the 3'-terminal CCA sequence of tRNA. The eubacterial, eukaryotic, and archaeal CCA-adding enzymes all share a single active-site signature motif, which identifies these enzymes as belonging to the nucleotidyltransferase superfamily. Here we show that mutations at Asp-53 or Asp-55 of the Sulfolobus shibatae signature sequence abolish addition of both C and A, demonstrating that a single active site is responsible for addition of both nucleotides. Mutations at Asp-106 (and to a lesser extent, at Glu-173 and Asp-215) selectively impaired addition of A, but not C. We have previously demonstrated that the tRNA acceptor stem remains fixed on the surface of the CCA-adding enzyme during C and A addition (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Taken together with this new evidence that there is a single active site for catalysis, our data suggest that specificity of nucleotide addition is determined by a process of collaborative templating: as the single active site catalyzes addition of each nucleotide, the growing 3'-end of the tRNA would progressively refold to create a binding pocket for addition of the next nucleotide.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024, USA.
authors.forename: D
authors.initials: D
authors.lastname: Yue
authors.forename: A M
authors.initials: AM
authors.lastname: Weiner
authors.forename: N
authors.initials: N
authors.lastname: Maizels
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism; D017931:DNA Primers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D002264:Carboxylic Acids; D017931:DNA Primers; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase