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Mechanism of ATP turnover inhibition in the EJC

Klaus H Nielsen1, Hala Chamieh, Christian B F Andersen

  • 1Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark.

RNA (New York, N.Y.)
|November 27, 2008
PubMed

Insights

The exon junction complex (EJC) locks onto RNA by stabilizing its conformation, not by inhibiting ATP hydrolysis. This mechanism prevents the release of components, revealing conserved features of ATP hydrolysis in DEAD-box helicases.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • The exon junction complex (EJC) is crucial for mRNA function after splicing.
  • The EJC core, comprising MAGOH, Y14, MLN51, and eIF4AIII, binds to spliced mRNAs.
  • Previous studies suggested MAGOH/Y14 inhibits eIF4AIII's ATP turnover, trapping the EJC on RNA.

Purpose of the Study:

  • To elucidate the mechanism by which MAGOH/Y14 locks the EJC onto RNA.
  • To determine the structural basis of ATP hydrolysis inhibition by MAGOH/Y14.
  • To investigate the transition state of ATP hydrolysis in DEAD-box helicases.

Main Methods:

  • Reconstitution and crystal structure determination of the EJC core bound to ADP-AIF(3).
  • Structural comparison with the EJC-ADPNP complex and other helicases.
  • Biochemical assays to demonstrate ATP hydrolysis and component release.

Main Results:

  • The crystal structure of the EJC core bound to ADP-AIF(3) reveals a dissociative transition state.
  • MAGOH/Y14 does not prevent ATP hydrolysis but locks the EJC conformation.
  • ATP hydrolysis occurs within the EJC, but release of products and RNA is blocked.
  • The ADP-AlF(3) method successfully reconstituted a transition state complex for Dbp5-RNA.

Conclusions:

  • MAGOH/Y14 locks the EJC onto RNA by preventing product and RNA release, not by inhibiting ATP hydrolysis.
  • The study reveals unifying features of ATP hydrolysis across different helicases.
  • ADP-AlF(3) is a valuable tool for studying DEAD-box ATPase transition states.

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