Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.
The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.
Toh
Yukimatsu
Y
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba-shi, Ibaraki, Japan.
Takeshita
Daijiro
D
Numata
Tomoyuki
T
Fukai
Shuya
S
Nureki
Osamu
O
Tomita
Kozo
K
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; C440000:A adding enzyme; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase