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.