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MECHANISM AND REGULATION OF
EUKARYOTIC PROTEIN SYNTHESIS
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Thomas E. Dever, PhD, Head,
Section on Protein Biosynthesis Pankaj Alone, PhD, Postdoctoral
Fellow M. Shamsul Arefin, PhD, Postdoctoral
Fellow Madhusudan Dey, PhD, Postdoctoral
Fellow Jeanne M. Fringer, PhD, Postdoctoral
Fellow Eun Joo Seo, PhD, Postdoctoral
Fellow Byung-Sik Shin, PhD, Postdoctoral
Fellow Chune Cao, Biological Laboratory
Technician Joo-Ran
Kim, BS, Special Volunteer
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We are
studying the mechanism and regulation of eukaryotic protein synthesis. A
chief interest is the role of GTPases in translation initiation. We have
shown that the universally conserved translation initiation factor eIF5B, an
ortholog of bacterial translation initiation factor IF2, promotes ribosomal
subunit joining in the second GTP-dependent step of the translation
initiation pathway. We are using mutational analyses to study the
structure/function properties of eIF5B, to determine the role of GTP binding
and hydrolysis by the factor, and to study its interaction with the ribosome.
We are also analyzing the structure-function properties of the factor eIF2,
which binds tRNAiMet to the ribosome, and we are
studying how eIF2 activity is downregulated by phosphorylation. Our studies
focus on the determinants of substrate recognition by the eIF2alpha protein
kinases and the structure and regulation of these enzymes. The human
eIF2alpha kinase PKR is an important component of the interferon-mediated
antiviral response. 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, which increase or
decrease inhibition, we are defining the molecular determinants of substrate
recognition in both eIF2alpha and PKR. Molecular,
biochemical, and structural analysis of eIF5B/IF2, a universally conserved
translation initiation GTPase Arefin, Cao, Fringer,
Kim, Shin, Dever; in collaboration with Burley, Lorsch, Pestova 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 and promotes the subunit-joining step of translation initiation.
Like bacterial IF2, eIF5B is a GTP-binding protein and possesses
ribosome-dependent GTPase activity. By switching the nucleotide specificity
of eIF5B, we demonstrated that two GTP molecules are required in eukaryotic
translation initiation as opposed to the single GTP requirement in bacterial
translation initiation (Lee et al., 2002). In collaboration with Stephen
Burley at The Rockefeller University, we determined the X-ray structure of an
archaeal form of eIF5B. The chalice-shaped protein consists of four domains:
the G domain and domains II and III form the cup of the chalice, a long alpha
helix forms the stem, and domain IV the base of the chalice. GTP binding to
eIF5B causes modest conformational changes in the G domain and cup of the
protein that are amplified by a lever-type mechanism (via the chalice stem
helix), resulting in significant movement of domain IV (the base of the
chalice). Our current efforts aim at elucidating the structure-function
properties of eIF5B and at a molecular dissection of the role of GTP binding
and hydrolysis by the factor. In contrast to the archaeal factor, yeast eIF5B
contains a long N-terminal extension. Deletion of this N-terminal region of
eIF5B has no impact on yeast cell growth; however, deletion of about 25
C-terminal residues of eIF5B slightly impairs yeast cell growth, and the
growth defect is exacerbated by deletion of the N-terminal region of eIF5B.
We propose that the growth defect reflects impaired ribosome binding because
the C-terminal domain IV of eIF5B binds to the factor eIF1A, which associates
with the small ribosomal subunit. Consistent with this hypothesis, the
C-terminal truncation of eIF5B impairs binding to eIF1A. To
assess the role of GTP binding and hydrolysis by eIF5B, we mutated the Switch
1 (Sw1) and Switch 2 (Sw2) motifs in the factor’s GTP-binding domain.
Mutation of a threonine conserved in Sw1 of all GTP-binding proteins
eliminated eIF5B GTPase and translational stimulatory activities and severely
impaired yeast cell growth but did not impair subunit joining. Intragenic
suppressors of the eIF5B Sw1 mutant restored yeast cell growth, but not eIF5B
GTPase activity. Biochemical studies revealed that the suppressor mutations
lowered the ribosome-binding affinity of the factor. The uncoupling of eIF5B
GTPase from translation-stimulatory activities suggests a regulatory rather
than mechanical role for eIF5B GTP hydrolysis in translation initiation. We
propose that a GTP-regulated switch governs the ribosome affinity of eIF5B.
In the presence of GTP, eIF5B binds to the ribosome, and following GTP
hydrolysis, the factor is released (Shin et al., 2002). Mutation of a
conserved glycine in Sw2 impaired eIF5B GTP-binding and hydrolysis activities
and yeast cell growth. Interestingly, an intragenic suppressor of the Sw2
mutation mapped to Sw1 and restored GTP binding and hydrolysis, a result that
provides the first genetic and biochemical evidence that the Sw1 and Sw2
elements in a G domain cooperate to promote GTP binding and hydrolysis. On
most mRNAs, ribosomes initiate translation at the AUG codon closest to the 5´
end. However, in yeast lacking eIF5B, or expressing eIF5B mutants that lack
GTPase activity, there was an increase in ribosomes’ leaky scanning,
bypassing the first AUG codon and instead initiating translation at a
downstream AUG codon. We propose that the GTPase activity of eIF5B serves as
a checkpoint to ensure efficient and accurate subunit joining during
translation initiation (Lee et al., 2002; Shin et al., 2002). In yeast, this
leaky scanning impairs expression of the GCN4 protein, and we are currently
screening for suppressor mutations that restore GCN4 expression in
cells lacking eIF5B. We propose the following model for the role of eIF5B in
translation initiation: a 40S ribosomal complex scans an mRNA and reaches an
AUG codon before GTP hydrolysis by eIF2 results in release of initiation
factors; binding of eIF5B•GTP to this complex stabilizes Met-tRNA
binding and promotes joining of the large ribosomal subunit; after 60S
joining, eIF5B hydrolyzes GTP, and the factor is released enabling the 80S
ribosome to begin translation elongation. Dever TE. Gene-specific regulation by general translation
factors. Cell 2002;108:545-556. Lee JH, Pestova TV, Shin B-S, Cao C, Choi SK, Dever TE.
Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic
translation initiation. Proc Natl Acad Sci USA 2002;99:16689-16694. Shin B-S, Maag D, Roll-Mecak A, Arefin MS, Sonenberg N, Dever TE. Eukaryotic translation initiation factors
and regulators. Curr Opin Struct Biol 2003;13:56-63. Structure-function
analysis of eIF2gamma, the GTP-binding subunit of the eIF2 complex Alone, Cao, Dever; in
collaboration with Burley The initiation
factor eIF2 is a three-subunit complex that delivers Met-tRNA to the ribosome
in an early step in the translation initiation pathway. The eIF2 binds to
GTP, and GTP-binding is required to form a stable
eIF2•GTP•Met-tRNA ternary complex. The gamma subunit of eIF2
contains the GTP-binding domain of the factor, and, recently, in
collaboration with Stephen Burley at The Rockefeller University and
Structural GenomiX, Inc., we obtained the crystal structure of eIF2gamma from
the archaeon Methanococcus jannaschii. Our structural data revealed a
striking similarity between the structures of eIF2gamma and the bacterial
translation elongation factor EF-Tu. The structural homology reflects the
functional similarity between eIF2 and EF-Tu, as EF-Tu delivers charged tRNAs
to the ribosome during translation elongation. Based on the structure of the
EF-Tu•GTP•Phe-tRNA complex, we predicted the site of the Met-tRNA
binding site on eIF2gamma. To test this model, we mutated residues in the
predicted tRNA binding site of yeast eIF2gamma and then examined the ability
of the mutants to support yeast cell growth. Mutation of the conserved
glycine at residue 397 (Gly-397) to alanine impaired cell growth, and,
importantly, this slow-growth phenotype was rescued by overexpressing
initiator Met-tRNA. Our data thus support the notion that Gly-397 is an
important determinant in the Met-tRNA binding site of eIF2. In addition to
mapping the tRNA binding site in eIF2gamma, we are examining the architecture
of the eIF2 complex by mutating the conserved surface residues on eIF2gamma.
Of particular note, mutation of aspartic acid at residue 403 (Asp-403) in
yeast eIF2gamma to alanine (D403A) impaired cell growth, with the growth
defect suppressed by overexpressing the alpha subunit of eIF2. This in
vivo result indicates that Asp-403 forms part of the binding interface
between the alpha and gamma subunits of eIF2, and biochemical analysis of the
corresponding mutations in archaeal eIF2gamma confirmed that the D403A
mutation disrupted the binding to eIF2alpha. Future experiments will seek to
map the eIF2beta binding site on eIF2gamma and further characterize the role
and mechanism of GTP hydrolysis by eIF2. Roll-Mecak A, Alone P, Cao C, Dever TE, Burley SK. X-ray
structure of translation initiation factor eIF2gamma: implications for tRNA
and eIF2alpha binding. J Biol Chem 2004; 279:10634-10642. Poxvirus
pseudosubstrate inhibitors of eIF2alpha kinases: molecular implications for
kinase-substrate interactions Seo, Cao, Dever; in
collaboration with Kennedy, Ramelot Phosphorylation
of the translation factor eIF2alpha on serine at residue 51 (Ser-51) by the
kinase PKR is an important component in the antiviral defense mechanism in
mammalian cells. Several poxviruses express small proteins resembling the
N-terminal third of eIF2alpha, and previous work demonstrated that the
vaccinia virus K3L protein is a pseudosubstrate inhibitor of the eIF2alpha
kinases. Previously, we showed that expression of either the vaccinia virus
K3L or the swinepox virus C8L protein reduced eIF2 alpha phosphorylation and
blocked the toxic effects associated with expression of PKR in yeast. The
inhibition of PKR was dependent on the sequence motif “KGYID,”
which is conserved among all K3L homologs and eIF2 alpha, and located around
30 residues C-terminal of the Ser-51 phosphorylation site in eIF2 alpha. We
collaborated with Theresa Ramelot and Michael Kennedy to characterize the K3L
homolog from myxoma virus, the M156R protein. The NMR structure of the M156R
protein revealed that the critical KGYID sequence motif and the region
corresponding to Ser-51 in eIF2alpha are located on the same face of the
M156R protein, likely forming the PKR recognition surface. In contrast to the
K3L protein, we demonstrated that the M156R protein competed with eIF2alpha
for phosphorylation by PKR. Thus, rather than functioning as a
pseudosubstrate inhibitor like the K3L protein, the M156R protein may
interfere with eIF2alpha phosphorylation through simple substrate competition
(Ramelot et al., 2002). To
gain further insights into pseudosubstrate inhibition of PKR, we are
employing two approaches. First, we are characterizing K3L mutants that are
more potent inhibitors of PKR. Expression of wild-type K3L partially
suppresses the toxic effects of PKR in yeast. We identified several
hyperactive K3L mutants that more effectively suppress PKR toxicity in yeast.
Second, we identified 12 mutations in PKR that confer resistance to
inhibition by K3L. The PKR mutants are toxic in yeast strains co-expressing
the K3L protein and likewise retain the ability to phosphorylate eIF2alpha
even in the presence of the viral pseudosubstrate. Further in vivo
studies revealed that the PKR mutants are not simply hyperactive but that
they are specifically resistant to inhibition by the K3L protein. Consistent
with this finding, the mutations cluster in the C-terminal lobe of the kinase
domain and, based on structural modeling, likely alter points of contact
between PKR and its pseudosubstrate (or substrate). Ongoing experiments seek
to determine how the mutations in PKR render the kinase less sensitive to
pseudosubstrate inhibition. Kazemi S, Papadopoulou S, Li S, Su Q, Wang S, Yoshimura A,
Matlashewski G, Dever TE, Koromilas AE. Control of eukaryotic translation
initiation factor 2alpha (eIF2alpha) phosphorylation by the human
papillomavirus type 18 E6 oncoprotein: implications for eIF2alpha-dependent
gene expression and cell death. Mol Cell Biol 2004;24:3415-3429. Ramelot TA, Cort JR, Yee AA, Liu F, Substrate
recognition by the eIF2alpha protein kinases Dey, Cao, Dever; in
collaboration with Sicheri In
addition to PKR, three other kinases phosphorylate eIF2alpha on Ser-51. These
stress-activated eIF2alpha kinases downregulate protein synthesis in stressed
cells. The kinase GCN2 is universally conserved in eukaryotes and
phosphorylates eIF2alpha under conditions of limiting amino acids. To gain
insights into the mechanism of substrate recognition by the eIF2alpha kinases
GCN2 and PKR, we generated N- and C-terminal truncation mutants of yeast
eIF2alpha. Residues 1 through 180 of eIF2alpha were necessary and sufficient
for efficient phosphorylation of Ser-51 both in vivo and in vitro.
Deletion of as few as 10 N- or C-terminal residues from the eIF2alpha
1–180 protein severely impaired phosphorylation of Ser-51. The results
demonstrate that kinase-substrate recognition for eIF2alpha phosphorylation
is not limited to a short consensus sequence element flanking the
phosphorylation site, as demonstrated for other kinases. Phosphorylation
of eIF2alpha impairs protein synthesis by converting eIF2 from a substrate to
an inhibitor of its guanine-nucleotide exchange factor eIF2B. To define
specifically the eIF2alpha determinants required for Ser-51 phosphorylation
and for translational regulation, we randomly mutated the conserved residues
in the N-terminal regional of yeast eIF2alpha, including each of the six
residues flanking Ser-51 plus the residues in the conserved KGYID motif
located at residues 79 through 83. Few substitutions in the residues flanking
Ser-51 impaired phosphorylation; however, many mutations at these residues
altered translational regulation. Thus, we propose that the eIF2alpha
residues flanking Ser-51 are critical for inhibition of eIF2B by
phosphorylated eIF2. Consistent with this proposal, several mutations in
these residues impaired the ability of phosphorylated eIF2alpha to bind to
eIF2B. Interestingly, several mutations in the KGYID approximately 20 Å from
the phosphorylation site significantly impaired phosphorylation of Ser-51. Of
particular note, substitution of The
identification of substrate recognition determinants 20 Å from Ser-51
suggests that residues remote from the PKR catalytic site are important for
substrate recognition. Multiple sequence alignments of eIF2alpha kinase
domains with other unrelated kinases identified 13 residues that are
preferentially conserved among eIF2 alpha kinases. We systematically mutated
these conserved residues in PKR and screened for mutations that impaired PKR
toxicity in yeast, indicating a defect in Ser-51 phosphorylation. Mutation of
Thr-487 and nearby Phe-495 abolished PKR toxicity in yeast and Ser-51
phosphorylation both in vivo and in vitro. In contrast, the PKR
mutations did not impair kinase autophosphorylation or nonspecific histone
phosphorylation. Thus, PKR residues Thr-487 and Phe-495 are important
determinants for specific substrate recognition. To our knowledge, this is
the first identification of kinase domain mutations that impair specific
substrate recognition. We propose that eIF2alpha kinase specificity is
mediated by interactions between conserved determinants remote from the
kinase catalytic site and the substrate phosphorylation site. COLLABORATORS Stephen K. Burley, PhD, Howard Hughes
Medical Institute, The Rockefeller University, New York, NY, and Structural
GenomiX, Inc., San Diego, CA Michael A. Kennedy, PhD, Jon R. Lorsch, PhD, The Tatyana V. Pestova, PhD, SUNY-HSC at Theresa A. Ramelot, PhD, Frank Sicheri, PhD, Samuel Lunenfeld
Research Institute,
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