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J Biol Chem, Vol. 274, Issue 42, 30297-30302, October 15, 1999
From the A series of mutations in the highly conserved
N153KMD156GTP-binding motif of the
Saccharomyces cerevisiae translation elongation factor 1A
(eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two
critical regulatory processes of translation elongation, guanine
nucleotide exchange and translational fidelity, were analyzed in
strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A
site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in
vitro. All three mutant proteins also show an increase in the
Km for GTP. An in vivo system lacking
the guanine nucleotide exchange factor eukaryotic elongation factor
1B The G-protein superfamily members are defined by highly conserved
sequence motifs and structural features and regulate a variety of
critical cellular processes (1). The ability of G-proteins to
transition between active and inactive forms based on whether GTP or
GDP is bound, respectively, allows them to function as a molecular
switch (2). This transition is modulated by accessory factors that
stimulate either GTP hydrolysis (GTPase-activating proteins) or guanine
nucleotide exchange (guanine nucleotide exchange factors
(GEFs)).1 These factors can
regulate the activity of the G-protein in response to molecular and
cellular signals. Many mutations have been isolated, particularly in
residues important for GTP binding, that affect the ability of the
G-protein to interact with GTPase-activating proteins and GEFs or
function as a target for the activities of these factors.
The eukaryotic translation elongation factor 1A (eEF1A) is a member of
the G-protein family. The prokaryotic homolog, EF1A, was the first
G-protein x-ray crystal structure solved (3). eEF1A is a GTPase whose
activity is stimulated by binding of aminoacyl-tRNA, ribosomes, and
most importantly the presence of a codon-anticodon match between the
aminoacyl-tRNA and the A site codon of the ribosome-bound mRNA.
Thus, the ribosome acts as a GTPase-activating protein for eEF1A.
Correspondingly, eEF1A requires a GEF, eEF1B (4). Only the eEF1B All G-proteins in the translation factor family share three well
conserved motifs, GXXXXGK, DXXG and
NKXD (6). Previous structural and mutational studies of the
Escherichia coli EF1A protein have supported the important
role of the N135KXD138 motif.
Structurally, this motif is important for binding and recognizing the
guanine ring (7). Substitutions of Lys136 result in a
dominant negative growth effect that is suppressed by overexpression of
the prokaryotic GEF EF1B (8). Genetic analysis of the
Lys136 mutant protein indicates that intragenic suppressors
of the dominant negative phenotype cluster in the G-domain of EF1A and
all result in reduced affinity for EF1B (9). Other dominant negative
mutations in Asn135 to Asp and Ile also appear to function
in a similar manner. A homologous mutation in S. cerevisiae
eEF1A, N153D, is no longer functional as the only form of the protein
(10) and also results in a conditional dominant negative growth
phenotype.2 Thus, this motif
is important in interacting with G-protein-associated factors. Double
mutants altering both Asn135 and Asp138 can
completely inactivate nucleotide binding by EF1A, consistent with
effects on affinity (11). Thus, the importance of these residues of the
motif element on nucleotide binding is clear. Mutational analysis of
the conserved Asp138 in EF1A results in altered nucleotide
specificity, resulting in a protein that now binds XTP with affinities
near the GTP binding affinity of the wild-type protein (12, 13). The
mutant protein does not affect translational fidelity in
vitro, because there appears to be no major change in nucleotide
affinity. The NKXD GTP-binding element is thus demonstrated
to play important roles in both the specificity and affinity of
nucleotide binding in G-proteins.
We have utilized the basis of knowledge available for the
NKXD GTP-binding element to study the effect of GTP binding
and hydrolysis by eEF1A on the efficiency and accuracy of translation in the eukaryotic organism S. cerevisiae. Three mutant forms
of the protein that alter Asn153, Asp156, and
both residues of the N153KMD156 element all
result in reduced affinity for GTP, and two correspondingly can
suppress the requirement for catalyzed nucleotide exchange utilizing a
genetic system deficient in the GEF eEF1B Strains and Media--
E. coli DH5 DNA Manipulations--
Restriction endonucleases and DNA
modifying enzymes were obtained from Roche Molecular Biochemicals.
Construction of the tef2 alleles on
TRP1-based plasmids are described in Cavallius and Merrick
(10). The URA3-based plasmids were prepared by digesting the
TRP1-based pRS314-JCX plasmids containing the indicated
mutation: D156N (JC6, tef2-17), N153T/D156E (JC32,
tef2-18), or N153T (JC5, tef2-19)
with BamHI and ScaI and cloning the resulting
fragment into pRS316 (URA3 CEN) digested with
BamHI and ScaI. The resulting plasmids were
pTKB293 (tef2-17, D156N), pTKB300
(tef2-18, N153T/D156E), and pTKB299
(tef2-19, N153T).
Western Blot Analysis--
Yeast strains containing wild-type
eEF1A (MC214) or one of the three mutant forms of eEF1A: N153T
(TKY226), D156N (TKY228), and N153T/D156E (TKY229) were grown in liquid
YEPD to an A600 in mid-log phase (0.4 to 1.0 units) and extracts prepared by glass bead lysis. Approximately 0.5 µg of total protein, as determined by Bradford protein analysis
(Bio-Rad) were separated by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. Membranes were probed with
polyclonal antibodies to yeast eEF1A (1:5000 dilution) and Rfa1p
(1:1000 dilution, kindly provided by Dr. Steven Brill, Rutgers
University) and colorometrically detected for multiple time points by a
secondary antibody conjugated to alkaline phosphatase (1:10,000
dilution, Bio-Rad) to assure a linear response.
Temperature Sensitivity, Translational Fidelity, and Growth of
tef2 Strains--
Temperature and cold sensitivity were assayed
by growing a strain containing wild-type eEF1A (MC213) or a mutant form
of eEF1A as the only form of the protein: D156N (TKY278), N153T
(TKY280), and N153T/D156E (TKY282) in YEPD to an
A600 of 1.0. Serial 10-fold dilutions (5 µl
each) were spotted on YEPD followed by incubation at 13, 23, 30, and
37 °C for 3-7 days. Phenotypic suppression of the
lys2-801 (UAG) mutation was determined by spotting 10 µl of the same dilutions onto complete medium or complete medium lacking
lysine and incubating for 2-5 days at 30 °C. Paromomycin-induced misreading was similarly assayed on complete medium or medium lacking
lysine containing 0.5 mg/ml paromomycin. Strains containing a
tef5::TRP1 null allele and supported for growth by
the plasmid-borne TEF5 (JWY4229), an eEF1B Drug Sensitivity--
2-ml cultures of each strain were grown at
30 °C in YEPD to mid-log phase. At least two independent colonies
were assayed for each mutant allele tested. For each culture 0.3 ml was
spread plated onto YEPD plates, and 10 µl of each drug were pipetted onto sterile BBL 1/4-inch diameter-paper discs. The
concentrations of drugs used were 1 mM cycloheximide, 25 mM hygromycin B, and 0.4 mM paromomycin. A
maximum of two filters were placed on each plate, and the plates were
incubated for 2-3 days at 30 °C. Sensitivity to each drug was
measured by the radius of growth inhibition in mm around each disc.
Nonsense Suppression Assays--
Nonsense suppression assays
were performed on strains containing wild type (MC214) or one of the
mutant forms of eEF1A: N153T (TKY226), D156W (TKY228), and N153T/D156E
(TKY229) and the URA3 wild-type lacZ control
plasmid pUKC815tail (lacZ under the PGK1 promoter
with the PGK1 transcriptional terminator) or URA3
plasmids with in-frame nonsense codons in lacZ; pUKC819tail
(UGA), pUKC817tail (UAA), and pUKC818tail (UAG). The mutant and
wild-type strains containing each plasmid were grown overnight at
30 °C in medium lacking uracil to mid-log phase. At least four
samples for each strain, and plasmids were analyzed in duplicate using
the 2-nitrophenyl- In Vivo [35S]Methionine Incorporation--
Strains
containing wild-type eEF1A (MC213) or the mutant forms D156N (TKY278),
N153T (TKY280), and N153T/D156E (TKY282) were converted to
MET2 by transformation of a polymerase chain reaction fragment containing the wild-type MET2 gene and homologous
recombination. Liquid cultures (100 ml) were grown in medium lacking
methionine at 30 °C to an A600 of 0.5-0.7.
At the zero time point, 50 µM cold methionine and
[35S]methionine to a final concentration of 1 µCi/ml
(7.9 mCi/ml, 293.0 MBq/ml, NEN Life Science Products) were added to
each culture. At 10-min intervals the optical density
(A600) of the cultures were determined, and 1-ml
aliquots were removed to monitor labeled methionine incorporation by
cold trichloroacetic acid precipitation. Ice-cold 50% trichloroacetic
acid (0.2 ml) was added to each aliquot, and aliquots were incubated on
ice for 10 min, heated to 70 °C for 20 min, and filtered through
Whatman GF/C filters. Filters were washed with 10 ml of 5%
trichloroacetic acid (4 °C) and 10 ml of 95% ethanol, dried, and
counted in a scintillation counter. All time points were analyzed in triplicate.
Mutations were targeted to the N153KMD156
GTP-binding consensus element of yeast eEF1A in an attempt to alter the
nucleotide specificity (10). Based on homology to other G-proteins that
had successfully been modified for their nucleotide specificity (12,
20-22), as well as the pattern of hydrogen bonds and salt bridges
between the GTP moiety and eEF1A, a series of substitutions were
prepared in Asn153 and Asp156 (10). Three of
the resulting mutants were utilized in a series of in vivo
studies to correlate in vitro changes in eEF1A function with
in vivo growth and translation effects. These mutant alleles alter the first and last residues of the
N153KMD156 element from Asp156 to
Asn (tef2-17), Asn153 to Thr and
Asp156 to Glu (tef2-18), and
Asn153 to Thr (tef2-19). The mutant
proteins all show increased misincorporation in an in vitro
translation assay (10). By utilizing monitors of altered translation
and fidelity, we initiated a study to determine the consequences of
these mutants in vivo.
Because overexpression of eEF1A can result in changes in cell growth
(18) and fidelity,3 Western
blot analysis was performed to rule out the possibility that the
N153KMD156 mutations in eEF1A might result in a
enhanced stability or expression. Strains containing chromosomal
disruptions of both eEF1A genes (tef1 Further analysis of the effects of mutations in the NKXD
element of yeast eEF1A indicates that these mutations are not favorable for some aspects of translation, although they do support wild-type growth as the only form of the protein. Initial analysis of the sensitivity of strains expressing either wild-type eEF1A or one of the
three mutations to translation inhibitors indicates only a slight
increase in sensitivity to cycloheximide and little or no increase in
sensitivity to hygromycin B (Table II).
However, all three strains alter sensitivity to paromomycin, either
increasing (D156N) or reducing (N153T and N153T/D156E) sensitivity to
the compound. This result indicates that these strains may have altered translational fidelity (23, 24). In vitro analysis of the purified mutant proteins indicated that all three increase
misincorporation of leucine in the poly(U)-directed polyphenylalanine
synthesis assay (10). To determine whether changes in A site fidelity also occurred in vivo, we utilized quantitative reporter
constructs to assay for nonsense suppression. Using in-frame
lacZ constructs containing one of the three stop codons,
nonsense suppression was monitored relative to expression of the
wild-type lacZ protein. Fig.
2A shows that all three
mutants increase in the level of There are several mechanisms that might cause the reduced fidelity at
the A site conferred by these mutations. One mechanism in
vivo is an increase in the speed of translation. As shown in Fig.
1, this increase would not be due to increased eEF1A protein but would
have to result in a change in the activity of the protein. To monitor a
general increase in protein synthesis, total methionine incorporation
was determined for strains containing either wild-type eEF1A or one of
the three NKXD mutant forms of the proteins. Fig. 3 (A and B)
indicate that no change in total translation occurs in strains
containing the D156N or N153T mutations. The N153T/D156E mutant strain,
however, shows a statistically significant increase in the rate of
incorporation. (Fig. 3C).
Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor
1A Reduce Both Translational Fidelity and the Requirement for
Nucleotide Exchange*
,,¶
Department of Molecular Genetics and
Microbiology,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(eEF1B) and supported for growth by excess eEF1A was used to
show the two mutations with the highest Km for GTP
restore most but not all growth defects found in these eEF1B
deficient-strains to near wild type. An increase in
Km alone, however, is not sufficient for
suppression and may indicate eEF1B performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine
nucleotide exchange may not effectively perform all the functions of
eEF1A in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit is required for nucleotide exchange, and in the yeast
Saccharomyces cerevisiae eEF1B, like eEF1A, is an
essential gene product (5). The regulation of eEF1A activity by
GTPase-activating proteins and GEFs is critically important in
efficient and accurate protein synthesis and consequently cell growth.
(14). However, not all of
the phenotypes associated with the loss of the GEF are suppressed, and
reduced affinity for guanine nucleotide alone is not sufficient for
suppression. We further find that mutations that correspond to changes
previously characterized in the prokaryotic homolog EF1A result in
significant differences in function. The D156N mutation of yeast eEF1A,
unlike the EF1A mutant (13), results in a dramatic reduction in
translational fidelity. In fact all the yeast NKXD mutant
proteins and strains analyzed result in reduced A site fidelity as
monitored by misincorporation and nonsense suppression. The
NKXD GTP-binding motif provides a valuable resource for
understanding two important aspects of regulation of eEF1A activity,
translational fidelity and guanine nucleotide exchange.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used for
plasmid preparation. S. cerevisiae strains used in these
studies are listed in Table I. Standard yeast genetic methods were employed (15). Yeast cells were grown in
either YEPD (1% Bacto yeast extract, 2% peptone, 2% dextrose) or
defined synthetic complete medium (C or C
) supplemented
with 2% dextrose as a carbon source. Yeast were transformed by the
lithium acetate method (16). Strains supported for growth by
plasmid-borne eEF1A genes (TEF1 or TEF2) are
derivatives of MC213 or MC214 prepared by plasmid shuffling (17).
Strains deficient in eEF1B are derivatives of the heterozygous
TEF5/tef5::TRP1 diploid JWY4175 (14) and were
prepared by either transformation of a URA3-based plasmid
containing the indicated tef2 allele into JWY4175
followed by sporulation and dissection or by transformation into the
haploid JWY4229 (tef5::TRP1 pTEF5-LEU2) (18)
followed by spontaneous loss of the TEF5 LEU2 helper
plasmid.
Saccharomyces cerevisiae strains used
-deficient
strain suppressed by wild-type eEF1A (TKY283), or an eEF1B-deficient
strain suppressed one of the three eEF1A mutants D156N (TKY279),
N153T/D156E (TKY287), or N153T (TKY302) were assayed for growth and
fidelity defects as described above. Doubling times of the
eEF1B-deficient strains and the eEF1B-containing parental strain
were determined by measuring the growth in liquid culture of at least
two independent isolates of each strain. Cultures grown for 1 day in
YEPD at 30 °C were diluted to an A600 of
approximately 0.1 in fresh YEPD and grown at 30 °C with vigorous
shaking. Optical density (A600) was assayed approximately every 2 h. Cultures were diluted into fresh YEPD when the A600 reached mid-log phase (0.4-0.6
units) to allow continued monitoring.
-D-galactopyranoside assay previously described
(19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
tef2) were prepared with either plasmid-borne
wild-type TEF2 (MC213) or a mutant form of eEF1A,
tef2-17 (D156N, TKY278), tef2-19
(N153T, TKY280), or tef2-18 (N153T/D156E, TKY282).
Yeast grow at essentially wild-type rates with one copy of an eEF1A gene, and the three mutants are all functional as the only form of the
protein and have no dramatic effects on cell growth (10). Fig.
1 shows that when equal amounts of
protein extract are examined, there is no difference in the level of
protein between wild-type eEF1A and any of the mutant forms.
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Fig. 1.
Mutations in the NKXD
GTP-binding element of eEF1A do not affect the stability or level of
the protein. Strains expressing only the plasmid-borne wild-type
TEF2 (MC213) or a mutant allele encoding eEF1A,
tef2-17 (D156N, TKY278), tef2-19
(N153T, TKY280), or tef2-18 (N153T/D156E, TKY282)
were grown to mid-log phase at 30 °C, and total proteins were
extracted. Equal amounts of protein, as determined by Bradford assay,
were run on a Laemlli gel, transferred to nitrocellulose, and probed
with polyclonal antibodies to Rfa1p (as a loading standard, top
panel) and yeast eEF1A (bottom panel).
-galactosidase activity in a strain
containing a nonsense reporter construct. When expressed as the fold
wild type read through level for each stop codon (Fig. 2B),
it is clear that all three mutations are omnipotent suppressors.
Drug sensitivities of strains containing mutations in the NKXD
GTP-binding element of yeast eEF1A
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Fig. 2.
Strains containing mutations in the
NKXD GTP-binding element of eEF1A show increased
suppression of all three stop codons. Strains containing wild-type
(MC214) or one of the mutant forms eEF1A, tef2-17
(D156N, TKY225), tef2-18 (N153T/D156E, TKY229), and
tef2-19 (N153T, TKY226), and the URA3
wild-type lacZ control plasmid pUKC815tail or a
URA3 plasmid with an in-frame nonsense codon in
lacZ; pUKC819tail (UGA), pUKC817tail (UAA), and pUKC818tail
(UAG) were assayed for -galactosidase production by a
2-nitrophenyl--D-galactopyranoside assay. A
shows the -galactosidase units, and B indicates the fold
readthrough of each stop codon relative to the strain expressing
wild-type eEF1A.
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Fig. 3.
Strains containing a mutation in the
NKXD GTP-binding element show either no effect or a
slight increase in total protein synthesis. Strains expressing
only the plasmid-borne wild type (MC213, 
) or a mutant form of
eEF1A: A, tef2-17 (D156N, TKY278, 
);
B, tef2-19 (N153T, TKY280, 
);
C, tef2-18 (N153T/D156E, TKY282, 
) were
grown in medium lacking methionine at 30 °C to an
A600 of 0.5-0.7. 50 mM cold
methionine and 1 mCi/ml [35S]methionine (7.9 mCi/ml,
293.0 MBq/ml, NEN Life Science Products) were added to each culture,
and both the A600 and cold trichloroacetic acid
precipitable radioactivity were determined at each time point. The
graphs express the cpm/A600 at the indicated
time point (tx) over the
cpm/A600 at t0.
Because these three mutants support growth even though they show
molecular defects in vitro, we were interested in using
these mutations to test the hypothesis that mutant forms of eEF1A with reduced nucleotide affinity could suppress the requirement for guanine
nucleotide exchange in vivo. This hypothesis is based on our
previous finding that although yeast normally require the guanine
nucleotide exchange factor eEF1B, encoded by the TEF5 gene, the presence of a third copy of an eEF1A gene (either
TEF1 or TEF2) allows viability (14). Further,
some mutant forms of eEF1A more effectively suppress the requirement
for eEF1B. Biochemical analysis indicates that all three mutations
increase the Km for GTP from 0.14 µM
for wild-type eEF1A compared with 13.1 µM for D156N, 10.3 µM for N153T/D156E, and 6.0 µM for N153T
(10). We hypothesized that although all three mutant forms of eEF1A show a dramatic increase in the Km for nucleotide,
these levels are still below the cellular content of GTP (100 s of
µM). Thus, the effect of these mutations to reduce GDP
binding would be more dramatic in vivo. This change would
reduce the requirement for eEF1B and suppress some of the negative
growth effects normally seen when the requirement for eEF1B is
suppressed by an extra copy of a wild-type eEF1A gene (14).
Plasmids containing a wild-type eEF1A gene or one of the three mutants
were prepared on a URA3-based low copy plasmid. The plasmids
were placed into a strain of yeast with the chromosomal eEF1B gene
deleted (tef5::TRP1) where viability was
maintained by the presence of a TEF5 LEU2 plasmid. Following
addition of an eEF1A-encoding plasmid, the TEF5 LEU2 plasmid
was spontaneously lost following growth in medium lacking uracil.
Alternatively, the plasmids were transformed into a heterozygous
TEF5/tef5::TRP1 diploid strain (JWY4229) without
any helper plasmid, and only viable Trp+ spores that were also Ura+
were obtained, indicating the eEF1A-containing plasmid supported
viability. The resulting tef5::TRP1 strains
supported by the tef2-17 allele (D156N, TKY279), tef2-18 allele (N153T/D156E, TKY287),
tef2-19 allele (N153T, TKY302), a wild-type
TEF2 gene (TKY283), or the parental strain supported by the
eEF1B gene (TEF5, JWY4229) were assayed for growth
defects. The doubling time at 30 °C for an eEF1B-deficient strain
containing the D156N or N153T/D156E mutations are 2.20 and 2.25 h,
respectively. This growth is near that of a strain containing eEF1B
(2.04 h). An eEF1B-deficient strain containing the N153T mutation
showed poor suppression of the eEF1B requirement (Fig.
4, third panel) and a doubling
time of 3.7 h, higher than the 3 h for a strain suppressed by
wild-type eEF1A. Thus two mutants with the highest Km values for GTP (13.1 µM for D156N
and 10.3 µM for N153T/D156E), as derived from poly(U)
assays using 15 pmol of eEF1A and an Eadie-Hofstee plot (10), best
suppress the requirement for eEF1B.
|
Other characteristics of eEF1B-deficient strains suppressed by
excess eEF1A are cold- and temperature-sensitive growth and sensitivity
to translational inhibitors. The D156N and N153T/D156E mutants very
effectively suppress the cold-sensitive defect of a strain lacking
eEF1B, clearly better than wild-type eEF1A or the N153T mutation,
the latter of which is dead at 13 °C (Fig. 4, first
panel). eEF1B-deficient strains suppressed by wild-type eEF1A
show dramatic increases in sensitivity to translation elongation inhibitors, such as paromomycin, from 0 to 6 mm of inhibition of growth
in a lawn growth assay (Table III). The
D156N and N153T/D156E mutants not only suppress the growth defects at
30 °C but also restore the drug sensitivity of the
eEF1B-deficient strain to at or near wild-type levels (Table III).
Interestingly, all four strains lacking eEF1B show a dramatic
temperature-sensitive growth defect; in fact little to no difference is
seen between mutants that suppress the requirement for eEF1B well at
30 °C compared with the N153T mutant that shows very poor
suppression (Fig. 4, fourth panel). Thus, of the multiple
phenotypes associated with the loss of eEF1B activity in the cell,
mutations in eEF1A can suppress some but not all of these defects.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The NKXD GTP-binding consensus element is a hallmark of all G-proteins and a site of much of the nucleotide specificity of G-proteins. The three mutations of N153T, D156N, and N153T/D156E all retained the ability to bind GTP, although with a much higher Km (10). These mutants allow for the in vivo analysis of the effect of reduced nucleotide binding on the requirement for guanine nucleotide exchange. Additionally, because the cellular concentration of GTP is hundreds of micromolar, the Km for the three mutant forms of eEF1A described are all still an order of magnitude less than the physiological concentration of GTP (25). Thus, these mutants remain functional as the only form of the protein. The function, however, may allow for growth but does not necessarily prevent phenotypes associated with altered translation. These mutants also address the effect of changes in guanine nucleotide binding or GTP-dependent reactions on accurate translation. Strains expressing one of the three mutant alleles show characteristic changes in sensitivity to translation inhibitors consistent with altered elongation, such as a slight increase in sensitivity to cycloheximide and changes in sensitivity to paromomycin. The latter phenotype correlates with altered translational fidelity (19, 23, 24, 26).
Biochemical analysis of these three mutant forms of yeast eEF1A
provided evidence that all three result in codon misreading using the
in vitro polyphenylalanine synthesis assay. Additional forms
of translational fidelity that are assessable in vivo
include nonsense suppression and programmed and nonprogrammed ribosomal frameshifting. Previously, mutations in yeast eEF1A have been demonstrated to affect frameshifting at nondirected (17) and directed
signals (19, 27). A strain expressing any one of the three mutant
alleles shows no change in the ability to retrotranspose the yeast
Ty1 element, indicative of no alteration in programmed +1
frameshifting (data not shown). To more closely address misreading at
the A site, as is monitored in the leucine misincorporation assay,
nonsense suppression was monitored for strains containing a single copy
plasmid expressing wild-type eEF1A or one of the three mutations
(N153T, D156N, and N153T/D156E). All three mutant strains, while
showing normal growth rates, demonstrate increased readthrough of all
three stop codons (Fig. 2). These results demonstrate a correlation
between changes in translational fidelity in vitro and
in vivo. The small differences in the extent of the changes are not surprising because the in vitro system used does not
require the exchange factor eEF1B and other potential cellular
functions for eEF1A are not a complication. Thus, two A site events in
translational fidelity are similarly affected in these mutants, both of
which require the proper identification of the A site codon.
The question arises as to the molecular defect in eEF1A resulting in
the misreading. The N153T mutation shows a dramatic increase in the
intrinsic GTPase activity of eEF1A (from 0.6 to 2.8 pmol phosphate/pmol
protein), an effect consistent with eEF1A failing to wait for the
GTPase activation signal from the formation of a codon-anticodon pair.
The N153T/D156E double mutation shows a statistically significant
increase in total protein synthesis (Fig. 3). This is a 2-fold change,
but it should be noted that this change is relative to a strain
containing a single copy of the eEF1A gene and not the normal
complement of two chromosomal eEF1A genes (TEF1 and
TEF2). Thus, when the level of eEF1A is slightly lower than
normal, this effect may be more dramatic. Models of translational
fidelity and rates would predict that increasing the speed of
translation would reduce fidelity (28, 29). Consistent with this model,
we have also found that an eEF1B mutant strain with reduced total
translation shows enhanced translational fidelity (18).
The cause of the reduced translational fidelity by the D156N mutant is less apparent, although this mutation differs from the previous two in conferring sensitivity and not resistance to paromomycin. One possibility is that this mutant protein, which shows no difference in stimulation of the GTPase activity in the absence or presence of poly(U) with Phe-tRNA and ribosomes (9.0 pmol phosphate/pmol protein versus 9.3, respectively), is stimulating GTP hydrolysis in a ribosome-dependent manner but without sensing a codon-anticodon interaction. Alternatively, this mutation may affect a function of eEF1A not assayed, such as the affinity for the ribosome or actin binding. Guanine nucleotide binding to Dictyostelium eEF1A decreases the affinity for actin 7-7.5-fold (30), and thus the D156N mutant may alter the equilibrium between free and actin-bound eEF1A.
The altered nucleotide affinity of the three mutants also provides a
tool to dissect the requirement for catalyzed guanine nucleotide
exchange by the eEF1B subunit. The D156N and N153T/D156E mutants
share an alteration of Asp156 and the highest
Km value for GTP. Furthermore, an eEF1B-deficient strain containing either mutant grows at essentially the same rate at
30 °C as cells expressing wild-type eEF1B. These eEF1A mutations,
however, do not restore the temperature-sensitive defect of
an eEF1B-deficient strain (Fig. 4). Thus, further analysis of
mutations, either in eEF1A or other cellular factors, may help illuminate the nature of the temperature-sensitive defect of
eEF1B-deficient cells. Perhaps the remaining defect indicates that
there is another role for eEF1B. Alternatively, the level of eEF1A
expression or activity required for suppression of the requirement for
eEF1B may have a negative effect on the cell or one of the other
proposed functions of eEF1A such as actin binding (31). Suppression of the requirement for eEF1B is not seen for the N153T mutation. This
mutant has the lowest Km for GTP (other than the wild-type protein); thus there may be a threshold of affinity required
for efficient suppression of the need for catalyzed guanine nucleotide
exchange. Alternatively, this mutant also shows the lowest
Vmax (1.1 pmol of Phe compared with 2.4 pmol for
wild-type eEF1A), the highest misincorporation in vitro, and
the highest intrinsic GTPase. Thus, perhaps the suppression of the
requirement for eEF1B requires a form of eEF1A with the ability to
release GDP efficiently without compromising eEF1A activity. The
ability to dissect the function of a key G-protein such as eEF1A in a strain lacking a key regulatory factor such as the catalyzed guanine nucleotide exchange factor eEF1B is an excellent system to
understand G-protein regulation.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Stuart Peltz for providing reporter plasmids and Dr. Jonathan Dinman for comments on the manuscript.
| |
FOOTNOTES |
|---|
* This work was funded by National Institutes of Health Grant GM57483 (to T. G. K.) and a predoctoral fellowship from the New Jersey Affiliate of the American Heart Association (to E. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Microbiology, UMDNJ Robert Wood Johnson Medical School,
675 Hoes Ln., Piscataway, NJ 08854-5635. Tel.: 732-235-5450; Fax:
732-235-5223; E-mail: kinzytg@umdnj.edu.
2 A. L. Laitusis and T. G. Kinzy, unpublished observation.
3 N. Durko and T. G. Kinzy, unpublished observation.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
GEF, guanine
nucleotide exchange factor;
eEF1A, eukaryotic Elongation Factor 1A
(formerly EF-1);
eEF1B, eukaryotic Elongation Factor 1B
(formerly EF-1);
EF1A, Elongation Factor 1A (formerly EF-Tu);
EF1B, Elongation Factor 1B (formerly EF-Ts).
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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