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The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF(3), the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC-ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5-RNA-ADP-AlF(3), suggesting that the use of ADP-AlF(3) may be a valuable tool for examining DEAD-box ATPases in general.

The Sm and Sm-like proteins are conserved in all three domains of life and have emerged as important players in many different RNA-processing reactions. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. In marked contrast to eukaryotes, bacteria appear to contain only one distinct Sm-like protein belonging to the Hfq family of proteins. Similarly, there are generally only one or two subtypes of Sm-related proteins in archaea, but at least one archaeon, Methanococcus jannaschii, encodes a protein that is related to Hfq. This archaeon does not contain any gene encoding a conventional archaeal Sm-type protein, suggesting that Hfq proteins and archaeal Sm-homologs can complement each other functionally. Here, we report the functional characterization of M. jannaschii Hfq and its crystal structure at 2.5 A resolution. The protein forms a hexameric ring. The monomer fold, as well as the overall structure of the complex is similar to that found for the bacterial Hfq proteins. However, clear differences are seen in the charge distribution on the distal face of the ring, which is unusually negative in M. jannaschii Hfq. Moreover, owing to a very short N-terminal alpha-helix, the overall diameter of the archaeal Hfq hexamer is significantly smaller than its bacterial counterparts. Functional analysis reveals that Escherichia coli and M. jannaschii Hfqs display very similar biochemical and biological properties. It thus appears that the archaeal and bacterial Hfq proteins are largely functionally interchangeable.