Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation.
The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3' end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile beta-turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the beta-turn mutants reflect the consecutive conformations assumed by the beta-turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that beta-turn mutants can affect nucleotide selection because the growing 3' end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.
Cho
Hyundae D
HD
Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington 98195-7350, USA.
Verlinde
Christophe L
CL
Weiner
Alan M
AM
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001665:Binding Sites; D017124:Conserved Sequence; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021381:Protein Transport; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology
D019843:Archaeal Proteins; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase