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Mechanism and Regulation of Eukaryotic Protein Synthesis

Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
Cheryl G. Bolinger, PhD, Postdoctoral Fellow
Chune Cao, Biological Laboratory Technician
Madhusudan Dey, PhD, Research Fellow
Joo-Ran Kim, BS, Special Volunteer
Yvette R. Pittman, PhD, Postdoctoral Fellow
Stefan Rothenberg, PhD, Visiting Fellow
Preeti Saini, PhD, Visiting Fellow
Eun Joo Seo, PhD, Visiting Fellow
Byung-Sik Shin, PhD, Staff Scientist
Meghna Thakur, PhD, Visiting Fellow

We study the mechanism and regulation of protein synthesis, focusing on GTPases and protein kinases that control this fundamental cellular process. We use molecular-genetic and biochemical studies to dissect the structure-function properties of the translation initiation factors eIF2, a GTPase that binds methionyl-tRNA to the ribosome, and eIF5B, a second GTPase that catalyzes ribosomal subunit joining in the final step of translation initiation. Our studies have revealed a critical role for eIF2 in start codon selection and have defined a functionally important contact between eIF5B and the ribosome. We also investigate stress-responsive protein kinases that phosphorylate eIF2alpha. Recent studies revealed fast evolution of the antiviral kinase PKR in vertebrates and linked this to altered sensitivity to poxvirus inhibitors of the kinase. Finally, our studies on the factor eIF5A revealed an unanticipated role in the elongation phase of protein synthesis.

Structure-function analysis of eIF2gamma, the GTP and Met-tRNA binding subunit of the eIF2 complex

The translation initiation factor eIF2 is composed of three polypeptide chains that assemble to form a stable complex. The gamma subunit of eIF2 contains a consensus GTP-binding (G) domain, and the factor must bind to GTP to form a stable eIF2•GTP•Met-tRNA ternary complex. To gain a deeper understanding of the role of GTP binding and hydrolysis by eIF2, we mutated the conserved Asn135 residue in the eIF2gamma Switch I element to Asp. The N135D mutation impaired Met-tRNA binding to eIF2 and caused a Sui⁻ phenotype, enhancing initiation from a noncanonical UUG codon. Previous studies in the Donahue laboratory correlated a Sui⁻ phenotype with decreased Met-tRNA binding affinity, suggesting that premature release of Met-tRNA from eIF2 led to initiation at the UUG codon. Consistently, an A208V mutation restored Met-tRNA binding affinity and suppressed the slow-growth and Sui⁻ phenotypes of the eIF2gamma-N135D mutant. In contrast, an A382V mutation restored Met-tRNA binding and suppressed the slow-growth, but not the Sui⁻, phenotype. Moreover, an eIF2gamma-A219T mutation impaired Met-tRNA binding but unexpectedly enhanced the fidelity of initiation, suppressing the Sui⁻ phenotype associated with the eIF2gamma-N135D,A382V mutant. This uncoupling of start codon selection and Met-tRNA binding affinity to eIF2 indicates a more direct role for eIF2 in start site recognition. Interestingly, overexpression of eIF1, which is thought to monitor codon-anticodon interaction during translation initiation, likewise suppressed the Sui⁻ phenotype of the eIF2gamma mutants. We propose that structural alterations in eIF2gamma subtly alter the conformation of Met-tRNA on the 40S subunit and thereby affect the fidelity of start codon recognition independently of Met-tRNA binding affinity (1).

Structure-function analysis of the universally conserved translational GTPase eIF5B/IF2

In the final step of translation initiation, the large 60S ribosomal subunit joins with the 40S subunit, already bound to an mRNA, to form an 80S ribosome competent for protein synthesis. We previously discovered the translation initiation factor eIF5B, an ortholog of the bacterial translation factor IF2, and showed that it catalyzes ribosomal subunit joining. The GTPase binds to GTP and hydrolyzes the nucleotide in the presence of 80S ribosomes. Our current efforts aim to elucidate eIF5B’s structure-function properties and to understand the role played by eIF5B in GTP binding and hydrolysis.

The function of both eIF5B and the translational GTPases that promote elongation and termination of protein synthesis relies on proper interaction with the ribosome. While cryo-EM studies have revealed binding sites for the GTPases on the ribosome, in vivo data supporting these sites have not been reported. Our previous studies on an eIF5B mutant that was unable to hydrolyze GTP revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. Thus, mutations that impair GTP hydrolysis by the eIF5B impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. After identifying intragenic suppressor mutations of eIF5B GTPase-deficient mutants that restore cell growth by lowering the ribosome binding affinity of eIF5B, we reasoned that it should be possible to obtain mutations in the ribosome that likewise reduce eIF5B binding and suppress the toxic affects associated with expression of GTPase-defective mutants of eIF5B. An eIF5B-H480I mutation abolishes GTPase activity and causes a severe growth defect in yeast. We identified a mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B that suppress the toxic effects associated with expression of the eIF5B-H480I mutant in yeast. Both the rRNA and intragenic mutations lowered the ribosome binding affinity of eIF5B, indicating that the mutations enable eIF5B release from the ribosome in the absence of GTP hydrolysis. Linking the hydroxyl radical generator BABE at the sites of the domain II suppressors in eIF5B, we mapped the region of the ribosome contacted by domain II of eIF5B. Interestingly, the domain II suppressors contacted the body of the 40S subunit in the vicinity of helix h5. Thus, the rRNA and domain II suppressors affect the same contact surface between eIF5B and the 40S ribosomal subunit. Given that the helix h5 mutation also impairs translation elongation factor function, we propose that the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit (2).

Molecular analysis of eIF2alpha protein kinase activation, substrate recognition, and evolution

Phosphorylation of eIF2alpha is a common mechanism for downregulating protein synthesis under stress conditions. Four distinct kinases phosphorylate eIF2alpha on Ser51 under different cellular stress conditions. GCN2 responds to amino acid limitation, HRI to heme deprivation, PERK to ER stress, and PKR to viral infection. Consistent with their common activity to phosphorylate eIF2alpha on Ser51, the kinases show strong sequence similarity in their kinase domains. Phosphorylation of eIF2alpha converts eIF2 from a substrate to an inhibitor of its guanine-nucleotide exchange factor eIF2B. The inhibition of eIF2B impairs general translation, slowing the growth of yeast cells and, paradoxically, enhancing the translation of the GCN4 mRNA required for yeast cells to grow under amino acid starvation conditions. We used structural, molecular, and biochemical studies to define how the eIF2alpha kinases recognize their substrate.

In collaboration with Frank Sicheri, we obtained the X-ray structure of eIF2alpha bound to the catalytic domain of PKR. Back-to-back dimerization enables each PKR protomer to engage a molecule of eIF2alpha in the crystal structure. Given that all four eIF2alpha kinases share the PKR residues mediating kinase domain dimerization and eIF2alpha recognition, we propose that all four kinases similarly dimerize and recognize eIF2alpha. Based on our results, we propose an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation on Thr446, which in turn is required for specific eIF2alpha substrate recognition.

Phylogenetic analyses of the eIF2alpha kinases plus four unrelated protein kinases revealed fast evolution of the PKR kinase domain in vertebrates. These evolutionary studies also revealed evidence of positive diversifying selection at specific sites in the PKR kinase domain. Substitution of positively selected residues in human PKR with residues found in other species altered the sensitivity to PKR inhibitors from different poxviruses (3). A comparison of the sensitivity of human and mouse PKR to poxviral pseudosubstrate inhibitors revealed differences that were traced to positively selected residues near the eIF2alpha-binding site. Interestingly, 10 of 12 mutations identified in a genetic screen for PKR mutations conferring resistance to K3L inhibition occurred at sites that were under positive selection during evolution (4). Taken together, our results indicate how an antiviral protein (PKR) evolved to evade viral inhibition while maintaining its primary function (phosphorylation of eIF2alpha). Moreover, our identification of species-specific differences in PKR susceptibility to viral inhibitors has important implications for studying human infections in nonhuman model systems.

Poxvirus regulation of protein kinase PKR

As part of the mammalian cell's innate immune response, the double-stranded RNA–activated protein kinase PKR phosphorylates the translation initiation factor eIF2α to inhibit protein synthesis and thus block viral replication. To subvert this host cell defense mechanism, viruses produce inhibitors of PKR. Several members of the poxvirus family express two types of PKR inhibitor: a pseudosubstrate inhibitor and a double-stranded RNA–binding protein called E3L. The vaccinia virus K3L protein resembles the N-terminal third of eIF2alpha, with both proteins containing a beta-barrel fold of the OB-fold family. Whereas high-level expression of human PKR was toxic in yeast, co-expression of the vaccinia virus K3L protein or the related variola (smallpox) virus C3L protein abrogated such growth inhibition. We used this yeast assay to screen for PKR mutants resistant to K3L inhibition and identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain in the vicinity of the eIF2alpha binding site. The PKR mutations specifically conferred resistance to the K3L protein, but not to the E3L protein, both in yeast and in vitro. In vitro studies revealed that wild-type PKR and the PKR mutants phosphorylated eIF2alpha with the same kinetics; however, the mutant kinase was less sensitive to inhibition by K3L. Consistently, the PKR-D486V mutation led to a nearly 15-fold decrease in K3L binding affinity. Our results support the identification of the eIF2alpha binding site on an extensive face of the C-terminal lobe of the kinase domain and indicate that subtle changes in the PKR kinase domain can drastically affect pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate versus substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser51 phosphorylation site (4).

In related studies, we are identifying and characterizing PKR mutants resistant to inhibition by the E3L protein. These PKR mutations map to both the kinase domain dimerization interface and the N-terminal regulatory [double-stranded RNA–(dsRNA) binding] domain of the protein. Consistent with the different natures of the two poxvirus inhibitors of PKR, the PKR mutations that confer resistance to E3L do not confer resistance to K3L. The identification of E3L-resistant mutations in the PKR dimerization interface is consistent with the notion that E3L inhibits PKR by forming inactive heterodimers with the kinase. Future in vitro studies will directly examine the impact of the mutations on PKR dimerization and E3L inhibition.

Molecular analysis of the hypusine-containing protein eIF5A

The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine [N^e-(4-amino-2-hydroxybutyl)lysine], was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes, and to stimulate methionyl-puromycin synthesis. As eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular mRNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay and to nucleocytoplasmic transport. Using molecular-genetic and biochemical studies, we showed that eIF5A promotes translation elongation. Depletion of eIF5A or shifting a temperature-sensitive (ts-) eIF5A mutant to the non-permissive temperature resulted in the accumulation of polysomes, mimicking the affect of the translation elongation inhibitor cycloheximide. Moreover, inactivation of eIF5A increased the ribosomal transit time, the amount of time required, following initiation, for a ribosome to synthesize and release a completed protein. The translation elongation defect in extracts from the eIF5A ts- mutant strain was suppressed by addition of recombinant eIF5A from yeast, but not by a derivative lacking hypusine. Moreover, eIF5A enhanced the rate of tripeptide synthesis in reconstituted translation elongation assays. Finally, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin and impaired programmed ribosomal frameshifting. These results indicate that eIF5A might function together with eEF2 to promote ribosomal translocation. Given that eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor (5).

Additional Funding

Trans-NIH/FDA Intramural Biodefense Research Program Award (2008-2009, ongoing)

Publications

Alone PV, Cao C, Dever TE. Translation initiation factor 2gamma mutant alters start codon selection independent of Met-tRNA binding. Mol Cell Biol 2008 28:6877-6888.
Shin B-S, Kim J-R, Acker MG, Maher KN, Lorsch JR, Dever TE. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit. Mol Cell Biol 2009 29:808-821.
Rothenburg S, Seo EJ, Gibbs JS, Dever TE, Dittmar K. Rapid evolution of protein kinase PKR alters sensitivity to viral inhibitors. Nat Struct Mol Biol 2009 16:63-70.
Seo EJ, Liu F, Kawagishi-Kobayashi M, Ung TL, Cao C, Dar AC, Sicheri F, Dever TE. Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein. Proc Natl Acad Sci USA 2008 105:16894-16899.
Saini P, Eyler DE, Green R, Dever TE. Hypusine-containing protein eIF5A promotes translation elongation. Nature 2009 459:118-121.

Collaborators

Michael G. Acker, PhD, The Johns Hopkins University, Baltimore, MD
Arvin C. Dar, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada
Katharina Dittmar, PhD, SUNY-Buffalo, Buffalo, NY
Daniel E. Eyler, PhD, The Johns Hopkins University, Baltimore, MD
James S. Gibbs, PhD, NIAID, NIH, Bethesda, MD
Rachel Green, PhD, The Johns Hopkins University, Baltimore, MD
Jon R. Lorsch, PhD, The Johns Hopkins University, Baltimore, MD
Frank Sicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada

Contact

For more information, email tdever@box-t.nih.gov or visit spb.nichd.nih.gov

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