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

摘要

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-D73N74, mini-D73N74C75 and mini-D73C74N75; D73 is a discriminator nucleotide and N is either A, G, or U). The mini-D73N74 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-D₇₃C₇₄N₇₅ 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.