Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

CCA-adding enzyme builds the 3′-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D ₇₃N₇₄, mini-D₇₃N₇₄C₇₅ and mini-D₇₃C₇₄N₇₅; D₇₃ is a discriminator nucleotide and N is either A, G, or U). The mini-D ₇₃N₇₄ complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N₇₄ to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D₇₃C₇₄C₇₅ complex, the mini-D₇₃N₇₄C₇₅ and mini-D₇₃C ₇₄N₇₅ complexes adopt inactive open forms. Only the mini-D₇₃C₇₄U₇₅ accepts AMP to a similar extent as mini-D₇₃C₇₄C₇₅, and ATP shifts the enzyme to a closed, active form and allows U₇₅ to flip for AMP incorporation. These findings suggest that the 3′-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.