(Received for publication, March 7, 1995)
From the
Two elongation factors drive the ribosomal elongation cycle;
elongation factor 1 (EF-1
) mediates the binding of an
aminoacyl-tRNA to the ribosomal A site, whereas elongation factor 2
(EF-2) catalyzes the translocation reaction. Ribosomes from yeast and
other higher fungi require a third elongation factor (EF-3) which is
essential for the elongation process, but the step affected by EF-3 has
not yet been identified. Here we demonstrate that the first and the
third tRNA binding site (A and E sites, respectively) of yeast
ribosomes are reciprocally linked; if the A site is occupied the E site
has lost its binding capability, and vice versa, if the E site
is occupied the A site has a low affinity for tRNAs. EF-3 is essential
for EF-1
-dependent A site binding of aminoacyl-tRNA only when the
E site is occupied with a deacylated tRNA. The ATP-dependent activity
of EF-3 is required for the release of deacylated tRNA from the E site
during A site occupation.
Ribosomes from organisms of all kingdoms require two factors in
the course of the reactions of the elongation cycle; elongation factor
1 (EF-1
) (
)is responsible for the codon-dependent
selection of the cognate aminoacyl-tRNA at the ribosomal A site and
elongation factor 2 (EF-2) for the translocation reaction. The
discovery of a third elongation factor (EF-3) essential for the
elongation cycle in the yeast Saccharomyces cerevisiae was
therefore a surprise(1) . Furthermore, this factor is a
ribosome-dependent ATPase, which also accepts GTP and ITP as substrates
and to a lesser extent pyrimidine
nucleotides(2, 3, 4) . This wide substrate
specificity of EF-3 is one principal difference from other
translational factors, which show a stringent requirement for
GTP(5) .
EF-3 from S. cerevisiae consists of a
single polypeptide chain of 1044 amino acids(6, 7) .
The genes encoding the EF-3 analogues in Candida albicans and
in Pneumocystis carinii, higher fungi than yeast, have also
recently been isolated and sequenced (8, 9, 10) . The deduced amino acid sequences
from these two species show over 75% identity with the EF-3 from S.
cerevisiae. The Candida EF-3 gene could supplement the
disrupted EF-3 gene in S. cerevisiae, demonstrating the
functional identity of EF-3 among different fungi(9) . The
activity of EF-3 is distinct from that of EF-1 and EF-2, since
EF-1
and EF-2 from yeast can functionally replace their
counterparts in the presence of ribosomes from rat liver or wheat germ,
whereas an EF-3 dependence is only observed with yeast
ribosomes(11, 12) . This dependence follows the source
of 40 S subunits as suggested by experiments with hybrid
ribosomes(12) . A monoclonal antibody against EF-3 blocks in vitro translation of poly(U) as well as natural mRNA, and
evidence was obtained that EF-3 was associated with polysomes rather
than with ribosomal subunits and that the protein is required for every
cycle of the elongation of the polypeptide chain(13) .
Polyclonal antibodies did not cross-react with extracts from mammals
(liver and reticulocytes) or plants (wheat germ) and, accordingly, did
not affect the respective translational systems(12) .
The
actual step of the elongation cycle promoted by EF-3 has not been
identified. The factor is not involved in the nucleotide exchange
reactions of either EF-1 or EF-2(4) . Slight stimulations
of some other partial reactions of the elongation cycle including
peptide bond formation and translocation have been
reported(14, 15) . More detailed examinations revealed
that aminoacyl-tRNA binding to the A site was stimulated by EF-3 but
only in the presence of catalytic amounts of EF-1
(4) ,
which does not easily explain the absolute requirement found for EF-3 in vivo(7) .
The elongation factors EF-1, EF-2, and EF-3 from
yeast were purified as described (2, 20, 21) and were homogeneous according to
acrylamide gel electrophoresis (Fig. 1).
Figure 1:
Sodium dodecyl sulfate polyacrylamide
gel electrophoresis of purified yeast elongation factors. The protein
bands were stained with Coomassie Brilliant Blue. Purification
protocols are according to published
procedures(2, 20, 21) . Lanes1 and 4 are molecular weight markers; lane2 represents EF-3; lane3 is EF-2; and lane5 is EF-1. Standard molecular weight markers are
myosin (194,000),
-galcactosidase (116,000), phosphorylase b (94,000), bovine serum albumin (67,000), and ovalbumin
(45,000).
Figure 2:
Kinetics of ternary complex binding to
ribosomes in the post-translocational state and the effects of EF-3. ,
[
P]tRNA
(450
dpm/pmol);
,
[
C]Val-tRNA
(570 dpm/pmol);
, ternary complex
[
H]Phe-tRNA
. EF-1
GTP
(1800 dpm/pmol);
,
[
P]tRNA
; ▾,
[
C]Val-tRNA
;
,
[
H]Phe-tRNA
. POST,
ribosomal complex in the post-translocational state; PRE,
ribosomal complex in the pre-translocational
state.
A rigorous
test of this assumption concerning the active fraction of ribosomes can
be made with a heteropolymeric mRNA displaying different codons at the
various ribosomal sites. Therefore, we designed an mRNA of 46
nucleotides. An mRNA sequence of about this length is protected against
RNase attack by the ribosomes(25, 26) . The mRNA
contains in the middle two unique codons, AUG and UUC, for methionine
(M) and phenylalanine (F), respectively. The sequence of the MF-mRNA is
G(A
G)
A
-AUG-UUC-(A
G)
A
U,
which cannot form stable secondary structures (important for efficient
tRNA binding) and can be obtained in large amounts by run-off T7
transcription. In the presence of the MF-mRNA, 0.36 AcPhe-tRNAs could
be bound per 80 S ribosome (Table 1, Experiment 1), about 20%
less binding than obtained in the presence of poly(U) message (0.50
when AcPhe-tRNA was added in saturating amounts; (24) ). This
decreased efficiency is typical for direct binding of N-blocked aminoacyl-tRNA to the P site when the binding
depends on a codon not located at the 5`-end of the
message(18) . Of the total amount of bound AcPhe-tRNA, 81%
could react with puromycin and thus can be considered to reside at the
P site. However, when a deacylated tRNA
was
prebound, 0.5 AcPhe-tRNAs were found statistically bound per ribosome,
and this binding was almost exclusively at the ribosomal A site (90%; Table 1). Obviously, the tRNA
fixes the
mRNA with the AUG codon at the P site, and as a result, all of the
bound mRNA displayed the UUC codon at the A site. The binding value of
AcPhe-tRNA at the A site (0.5 per 80 S) found under saturating
conditions of the ligand was identical to the maximal binding values
observed in the poly(U)-dependent titration experiment (24) .
This finding verifies the assumption of the active fraction of 60
± 10%, which confirms the conclusion that yeast ribosomes have
three tRNA binding sites. There is no evidence for a fourth tRNA
binding site on yeast ribosomes as claimed for rabbit liver
ribosomes(27) .
AcPhe-tRNA appears in the P site without
prebound tRNA but appears almost exclusively
at the A site when tRNA
is prebound (Table 1, Experiments 1 and 2, respectively). It follows that all
ribosomes with AcPhe-tRNA at the A site must have deacylated
tRNA
at the adjacent P site. After
translocation, the fraction of bound tRNA
and
AcPhe-tRNA molecules did not change significantly, but now the
AcPhe-tRNA predominantly resides at the P site (77%) according to the
puromycin reaction. These findings also mean that all ribosomes with an
AcPhe-tRNA present at the P site carry tRNA
at the E site. The tRNA must be firmly bound at the E site, since
no tRNA was lost from the ribosome during the manipulations and
incubations necessary to carry out the translocation reaction (Table 1, line 3). These results functionally confirm the
existence of an E site on yeast ribosomes.
An experiment
corresponding to that of Table 1but in the presence of EF-3 and
ATP yielded the same data, and kinetics of the translocation reaction
were identical in the presence or absence of EF-3 (data not shown). We
conclude from this experiment that EF-3 does not influence nonenzymatic
binding of tRNA to either the P or the A site. Similarly, the peptidyl
transferase reaction as well as the EF-2GTP-dependent
translocation reactions were also not influenced by EF-3.
The
ribosomal state tested in the next experiment reflects the standard
situation of an A site binding during an elongation cycle, i.e. both P and E sites are occupied with tRNAs (post-translocational
state). This state was achieved by an EF-2-dependent translocation of
[C]Val-tRNA
from the A site to the
P site and tRNA
from the P site to the E
site. Before translocation, only 6% of the bound Val-tRNA reacted with
puromycin. However, after translocation, 69% of Val-tRNA became
puromycin-reactive, indicating that indeed the majority of ribosomes
carrying Val-tRNA were in the post-translocational state. The
corresponding A site occupation with [
H]Phe-tRNA
was termed A site binding of the e-type (e for elongation; see (28) ). EF-3 dramatically affected the enzymatic A site binding
of the e-type. In the absence of EF-3, the ternary complex containing
Phe-tRNA could hardly occupy the A site, whereas in the presence of
EF-3 a 5-fold stimulation of the A site occupation occurs reaching
normal binding values (0.08 and 0.38 Phe-tRNAs per 80 S, respectively,
see Table 2). The strong EF-3 effects seen with the A site
occupation of the e-type is largely dependent on the presence of
EF-1
within the ternary complex, since AcPhe-tRNA hardly bound to
the A site when the E site was occupied (data not shown).
Figure 3:
Chasing of E site bound tRNA: comparison
of two post-translocational complexes containing
[P]tRNA
or
[
P]tRNA
.
,
[
P]tRNA
(850
dpm/pmol);
,
Ac-[
C]Phe-tRNA
(1000 dpm/pmol);
, [
P]tRNA
(510 dpm/pmol). The chasing exclusively affected the deacylated
tRNA in the E site; the AcPhe-tRNA in the P site was not affected.
Chasing agents (10
over ribosomes) are as follows: tRNA
(solid bar),
[
P]tRNA
(openbar), and tRNA
(shadedbar). For further explanations see ``Experimental
Procedures.''
The
``tightness'' of the E site binding and the direct effect of
EF-3 on this site were tested in the following way. We assessed chasing
effects at the E site by adding nonlabeled tRNA or tRNA
in a 10 molar excess over the ribosomes.
The data presented in Fig. 3demonstrate that under all
conditions the chasing substrate cognate to the E site codon clearly
exceeded the effects seen with the near cognate tRNA. Noncognate
tRNA
(codon: ACC) had no effect in the absence of EF-3.
The striking dependence on the anticodon character of the chasing
substrate identifies codon-anticodon interaction as an important
determinant of the binding of deacylated tRNA to the E site. A second
important feature of the experiments shown in Fig. 3, A and B, is the strong effect induced by EF-3
ATP. The
factor facilitates the chasing by both cognate and near cognate tRNAs,
and now even the noncognate tRNA has a low but significant effect. The
EF-3 effect is pronounced and E site-specific, i.e the EF-3
effect is observed in the absence of aminoacyl-tRNA and EF-1
.
The results shown in Fig. 4demonstrate that the E site-specific effect of EF-3 requires hydrolysis of ATP. There is no EF-3-dependent release of deacylated tRNA from the E site in the absence of ATP or in the presence of AMPPNP, the noncleavable analogue of ATP.
Figure 4:
Chasing of the E site bound tRNA:
dependence on EF-3 and ATP. The post-translocational state was made as
in Fig. 3B but with limiting amounts of 14 µM GTP. For chasing, a 10 molar ratio of nonlabeled tRNA was added as well as EF-3 (1 pmol per 80 S) and, where indicated,
ATP (1 mM) or AMP-PNP (1.5 mM). After 5 min at 30
°C the binding was determined by a nitrocellulose filter-binding
assay.
The experimental data reveal that the three essential features of the allosteric three-site model for the elongation cycle (31) are also valid for the yeast ribosomes. 1) In addition to the A and P sites, a third site (E site) exists for the yeast ribosomes. 2) The E site is specific for deacylated tRNA and is reciprocally linked to the ribosomal A site, i.e. occupation of the A site reduces the affinity for tRNA of the E site and vice versa. 3) The deacylated tRNA at the E site undergoes codon-anticodon interaction. The allosteric three-site model was first derived from experimental data obtained with ribosomes of the eubacteria E. coli(18) and then demonstrated for archaebacterial ribosomes from Halobacterium halobium(32) , and as shown here, it is also valid for the eukaryotic yeast ribosomes. The model thus probably describes universal features of elongating ribosomes.
The possible effect of EF-3 on
ribosomal function was tested in different assays covering the complete
elongation cycle. The factor affected neither the translocation extent
and speed nor tRNA binding to programmed ribosomes containing no tRNA
or one tRNA bound to the P site. In contrast, the EF-1-dependent
binding of aminoacyl-tRNA to a ribosomal complex containing two tRNAs
bound to the P and E sites required the presence of EF-3 and ATP ( Table 2and Fig. 2). Additionally, a direct involvement of
EF-3 with the ribosomal E site function was demonstrated with the
factor-dependent increase of chasing a tRNA from this site (Fig. 3). The EF-3-induced hydrolysis of ATP was essential for
this effect (Fig. 4), which constitutes the first reported
activity of this factor in the absence of EF-1
.
The function of
EF-3 can be neatly explained in the frame of the allosteric three-site
model. EF-3 is essential for the A site occupation of the e-type. If
the reciprocal linkage between the A and E sites is a stringent feature
of the elongation cycle, a release of deacylated tRNA from the E site
is a prerequisite for A site binding. The observed EF-3 effects can
thus be reconciled as follows. Deacylated tRNA is tightly bound to the
E site; EF-3-dependent ATP hydrolysis is required to enable the release
of tRNA from this site by binding the cognate ternary complex to the A
site. The binding energy contributed by EF-1 is obviously
essential for this allosteric transition from the post- to the
pretranslocational state, since acylated tRNA cognate to the A site
codon cannot trigger this reaction in the absence of EF-1
or in
the presence of EF-3 alone.
In higher eukaryotes a ribosomal ATPase/GTPase has been described(33) . It is tempting to speculate that EF-3 has become an integral component of the ribosome and exerts its function as a ribosomal protein. In prokaryotic ribosomes the recently described second EF-Tu-dependent GTP hydrolysis required for the binding of cognate aminoacyl-tRNA to the A site (34, 35, 36) could fulfill a function analogous to that of the EF-3-dependent ATP hydrolysis. It is thus possible that the EF-3 function defined here for yeast ribosomes reflects a universal feature of protein biosynthesis.