We are studying the mechanism and regulation of eukaryotic protein synthesis. An overarching interest is the universally conserved translation initiation factor eIF5B, an ortholog of bacterial translation initiation factor IF2. We have shown that eIF5B promotes ribosomal subunit joining in the second GTP-dependent step of the translation initiation pathway. In addition, we obtained the X-ray structure of eIF5B and are studying the structure/function properties of this factor and its interaction with the ribosome. We are also analyzing how the function of the factor eIF2, which binds tRNAiMet to the ribosome, is down-regulated by phosphorylation. Our studies focus on the determinants of substrate recognition by the eIF2a protein kinases and on the structure and regulation of these enzymes. The human eIF2a kinase PKR is an important component of the interferon-mediated antiviral response; consequently, many viruses encode inhibitors of PKR. We have reconstituted in yeast cells the antagonism of PKR by pseudosubstrate inhibitors encoded by poxviruses. By identifying mutations in these inhibitory proteins, and others in PKR, that increase or decrease inhibition, we are identifying molecular determinants of substrate recognition in both eIF2a and PKR.

Molecular, Biochemical, and Structural Analysis of eIF5B/IF2, a Universally Conserved Translation Initiation Factor
Arefin, Cao, Kim, Shin, Dever; in collaboration with Burley, Pestova, Lorsch, Hellen
We previously discovered the translation initiation factor eIF5B and characterized the factor from both yeast (encoded by the FUN12 gene) and humans. The eIF5B is an ortholog of the bacterial translation initiation factor IF2. Working in collaboration with Tatyana Pestova and Christopher Hellen at SUNY-Brooklyn, we found that human eIF5B promotes the subunit-joining step of translation initiation. The eIF5B resembles the bacterial translation initiation factor IF2. Like IF2, eIF5B is a GTP-binding protein and possesses ribosome-dependent GTPase activity. In collaboration with Stephen Burley at The Rockefeller University, we determined, by analyzing X-ray structures, three states of eIF5B from the archaeon Methano-bacterium thermoautotrophicum: inactive (eIF5B•GDP), active [eIF5B•GDPNP (mimick-ing GTP)], and nucleotide-free. The extended protein is composed of four domains and resembles a chalice. Comparison of the structures of the active and inactive forms of eIF5B revealed that modest structural changes in the GTP-binding domain (in the cup of the chalice) are amplified by an articulated lever mechanism that results in significant movement of domain IV (the base of the chalice). The GTP-binding domain and domain II of eIF5B resemble the first two domains of the translation elongation factors EF-Tu and EF-G. Based on this structural conservation, we proposed that eIF5B binds near the GTPase-activating center of the ribosome, as has been shown for EF-Tu and EF-G (Roll-Mecak et al., 2000; Dever et al., 2001).

The identification of eIF5B was particularly intriguing because it had been believed that only a single GTP molecule was used during translation initiation. However, the translation initiation factor eIF2, which binds the initiator Met-tRNA to the small ribosomal subunit in the first step of the translation initiation pathway, is also a GTPase. Therefore, the discovery of eIF5B predicts that two GTP molecules will be consumed during translation initiation. To test this prediction, a mutant form of human eIF5B was generated that changed the nucleotide specificity of the factor from GTP to XTP. Ribosome-dependent nucleotide hydrolysis assays confirmed that the mutation eliminated GTPase activity but endowed the factor with XTPase activity. Consistent with this altered nucleotide specificity, the mutant protein catalyzed subunit joining in the presence of XTP but not GTP. These results demonstrate that two GTP molecules are required in eukaryotic translation initiation. We propose that the GTP used by eIF2 plays a role in proper selection of the AUG start codon, whereas the GTP consumed by eIF5B promotes ribosomal subunit joining.

To assess the role of GTP-binding and hydrolysis by eIF5B, we mutated the Switch 1 motif in the factor’s GTP-binding domain. Substitution by alanine of a threonine that is conserved in all GTP-binding proteins eliminated the eIF5B GTPase and translational stimulatory activities. In addition, expression of this mutant protein in yeast severely impaired cell growth. Interestingly, the mutant factor retained the ability to promote subunit joining. Thus, GTP hydrolysis by eIF5B is necessary for protein synthesis but not for subunit joining. We screened for intragenic suppressor mutations that restored the ability of the eIF5B Switch 1 mutant to promote yeast cell growth. One suppressor mutation mapped to the GTP-binding domain; however, the mutation did not restore the GTPase activity of the factor. Biochemical studies demonstrated that the suppressor mutation lowered the ribosome-binding affinity of eIF5B. Thus, we propose that GTP-binding and hydrolysis by eIF5B regulates the binding of the factor to the ribosome.

On most mRNAs, ribosomes initiate translation at the AUG codon closest to the 5' end. However, in yeast lacking eIF5B or in eIF5B mutants that lacked GTPase activity, the level of ribosomal leaky scanning increased, i.e., the ribosomes bypassed the first AUG codon and instead initiated translation at a downstream AUG codon. We propose that the GTPase activity of eIF5B serves as a checkpoint to ensure efficient subunit joining during translation initiation. Thus, when a scanning 40S ribosomal complex reaches an AUG codon, GTP hydrolysis by eIF2 results in release of initiation factors. We propose that eIF5B•GTP binds to this complex, stabilizes Met-tRNA binding, and promotes joining of the large ribosomal subunit. Upon 60S joining, eIF5B hydrolyzes GTP, and the factor is released, enabling the 80S ribosome to begin translation elongation.

Poxvirus Pseudosubstrate Inhibitors of eIF2a Kinases: Molecular Implications for Kinase-Substrate Interactions
Liu, Cao, Dever; in collaboration with Ramelot, Kennedy
Previously, we demonstrated that the vaccinia virus K3L protein and the swinepox virus C8L protein are pseudosubstrate inhibitors of PKR, the mammalian interferon-induced eIF2a kinase. Expression of either the K3L or C8L protein reduced eIF2a phosphorylation and blocked the toxic effects associated with expression of PKR in yeast. This inhibition of PKR by the K3L and C8L proteins was dependent on a sequence motif KGYID, which is conserved among all K3L homologs and eIF2a and located around 30 residues C-terminal of the Ser-51 phosphoryla-tion site in eIF2a. As part of a structural genomics initiative, Theresa Ramelot, a postdoctoral fellow in Michael Kennedy’s laboratory, obtained by NMR the structure of the K3L homolog from myxoma virus, the M156R protein. It is interesting that the critical KGYID sequence motif and the region corresponding to Ser-51 in eIF2a are located on the same face of the M156R protein, likely forming the PKR recognition surface. Working in collaboration with the Kennedy laboratory, we demonstrated that the M156R protein is an efficient in vitro substrate for phosphorylation by PKR. Mutational analyses indicated that the primary site of phosphorylation is a Tyr residue located in the position corresponding to Ser-51 in eIF2a. In addition, the M156R protein competed with eIF2a for phosphorylation by PKR. Thus, rather than functioning as a pseudosubstrate inhibitor like the K3L protein, the M156R protein may interfere with eIF2a phosphorylation through simple substrate competition (Ramelot et al., 2002).

To extend this project, we are characterizing K3L mutants that are more potent inhibitors of PKR. Whereas wild-type K3L partially suppressed the toxic effects of PKR in yeast, these hyperactive K3L proteins more fully suppress PKR toxicity in yeast. In addition, we identified PKR mutants that are resistant to inhibition by K3L while retaining the ability to phosphorylate eIF2a. By sequencing these PKR mutants, we identified 12 single amino acid changes that make PKR resistant to K3L inhibition. An interesting finding is that these mutations cluster in the Cterminal lobe of the kinase domain; based on structural modeling, they likely alter points of contact between PKR and its pseudosubstrate (or substrate). Currently, we are using genetic and biochemical assays to determine how the mutations in PKR render the kinase less sensitive to pseudosubstrate inhibition.

Substrate Recognition by the eIF2a Protein Kinases
Dey, Liu, Cao, Dever
To gain insights into the mechanism of substrate recognition by the eIF2a kinases GCN2 and PKR, we are defining the minimal eIF2a substrate that is efficiently phosphorylated. We expressed and tested deletion mutants of eIF2a both in vivo and in vitro for phospho-rylation on Ser-51. Residues 1-180 of eIF2a appear to be necessary for high-level phosphorylation. Deletion of as few as 10 amino- or carboxyl-terminal residues from the eIF2a 1-180 protein severely impaired phosphorylation of Ser-51. These results, combined with the results of our studies on the K3L protein, suggest that GCN2 and PKR recognize a large domain of eIF2a. In addition, our results demonstrate that kinase-substrate recognition for eIF2a phosphorylation is not limited to a short consensus sequence element flanking the phosphorylation site, as has been demonstrated for other kinases. We employed an in vivo screen to identify the residues in yeast eIF2a that are critical for translational regulation and phosphorylation of Ser-51 by the eIF2a kinases. Few substitutions in the residues flanking Ser-51 affect substrate recognition; however, many mutations at these residues alter translational regulation by the eIF2a kinases. In contrast, mutations at residues 79–83, which include the KGYID sequence element that is conserved among the K3L homologs and eIF2a, significantly impaired phosphorylation of Ser-51. Of particular note is that substitution of Ala for Asp-83 at 32 residues from the phosphorylation site (at Ser-51) completely blocked eIF2a phosphorylation by GCN2 and PKR both in vivo and in vitro. Such a critical requirement for specific residues in a substrate remote from the site of phosphorylation has not been reported for any other protein kinase. The unprecedented importance of remote sequences, combined with the ease of conducting in vivo analyses of kinase and substrate mutants, makes the yeast eIF2a system ideal for studying substrate recognition by protein kinase.