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

Yukimatsu Toh, Tomoyuki Numata, Kazunori Watanabe, Daijiro Takeshita, Osamu Nureki, Kozo Tomita

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1944-1952
Number of pages9
JournalEMBO Journal
Volume27
Issue number14
DOIs
Publication statusPublished - Jul 23 2008
Externally publishedYes

Fingerprint

Adenosine Monophosphate
Maintenance
Transfer RNA
Nucleotides
Cytidine Monophosphate
Cytidine Triphosphate
Discriminators
Enzymes
Nucleic Acids
Adenosine Triphosphate
RNA
tRNA nucleotidyltransferase

Keywords

  • CCA
  • Fidelity
  • Template-independent polymerase
  • tRNA

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme. / Toh, Yukimatsu; Numata, Tomoyuki; Watanabe, Kazunori; Takeshita, Daijiro; Nureki, Osamu; Tomita, Kozo.

In: EMBO Journal, Vol. 27, No. 14, 23.07.2008, p. 1944-1952.

Research output: Contribution to journalArticle

Toh, Yukimatsu ; Numata, Tomoyuki ; Watanabe, Kazunori ; Takeshita, Daijiro ; Nureki, Osamu ; Tomita, Kozo. / Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme. In: EMBO Journal. 2008 ; Vol. 27, No. 14. pp. 1944-1952.
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