 |
INTRODUCTION |
The first step of prokaryotic mRNA translation requires at
least 3 proteins known as the initiation factors
IF1,1 IF2, IF3, and a
complexed GTP molecule, in addition to
fMet-tRNAfMet (1). Of these three
proteins only IF2 has a GTP-binding domain. During 30 S initiation
complex formation, the initiation factors, GTP,
fMet-tRNAfMet, and mRNA are bound to
the small ribosomal subunit. In this context IF2 binds at least three
different ligands: GTP, the 30 S ribosomal subunit, and the initiator
tRNA (1-3). The functional 70 S initiation complex ready for
elongation contains only the initiator tRNA and mRNA. During its
formation the initiation factors are released and GTP is hydrolyzed.
The presence and hydrolysis of GTP which are directly linked to the
presence of IF2 have been shown to be essential for initiation of
protein biosynthesis (4). However, the precise role of GTP is still not
clear. IF2 is one of the largest G-proteins known, and, in contrast to
other proteins of this class which carry the G-domain in their
N-terminal region, the G-domain of IF2 is centrally located in the
protein. IF2 has been shown to bind GTP with a 10-fold lower affinity
than GDP, in contrast to EF-Tu which binds GTP 100 times less
efficiently than GDP (5, 6). Its lower overall affinity for guanine nucleotides (1000-fold for GDP, 10-fold for GTP) might enable IF2 to
self-cycle from the GDP to the GTP state. Free IF2 does not show any
GTPase activity; this is induced upon binding of the 50 S subunit
during 70 S complex formation. The IF2 GTPase depends completely on the
presence of the ribosome (7, 8). However, in Bacillus
stearothermophilus, both IF2 and its isolated G-domain were shown
to be capable of hydrolyzing GTP in the absence of ribosomes when 20%
ethanol was included in the reaction (9).
In order to define the E. coli IF2 G-domain more precisely,
we had previously mutated very conserved residues suspected to be
involved in GTP binding or hydrolysis and assessed the effect of these
mutant proteins in vivo in a strain carrying a null mutation in the chromosomal copy of infB (4). The different mutations globally confirmed a theoretical three-dimensional model of the IF2
G-domain (10) and demonstrated the crucial importance of GTP hydrolysis
in translation initiation. Six out of seven mutations in that study
were lethal and of those, two (Val400 to Gly and
His448 to Glu) exhibited a strong dominant inhibitory
effect over the wild-type protein (4).
An alignment of the primary structures of several G-domains reveals
that the valine 400 residue of IF2 is equivalent to valine at position
20 in E. coli EF-Tu and, from a structural point of view,
corresponds to glycine at position 12 in the mammalian protooncogenic Ha-ras protein, p21. Indeed, these residues are found within
the G1 region (GXXXXGK), a consensus element of the G-domain
specifically involved in the interaction with the
and
phosphates of GDP/GTP (11-13). A V20G substitution in E. coli EF-Tu (analogous to the V400G mutation in IF2) causes a
5-10-fold reduction in GTPase activity and impairs its stimulation by
the ribosome (14, 15). The histidine 448 residue of IF2, the last
residue of the second consensus element (DXXGH) constituting
the G-domain, is well conserved in initiation and elongation factors of
different organisms (16, 17). Mutations of the corresponding residue of
EF-Tu (His84) underline the importance of this residue for
polypeptide synthesis and GTP hydrolysis (18, 19) but it is not yet
clear how this residue, which is not in direct contact with the
-phosphate of GTP, participates in GTP hydrolysis (20).
The work presented here follows up on our in vivo studies
and describes the major biochemical and enzymatic characteristics of
the V400G and H448E IF2 mutants in vitro. Based on their
impaired GTPase activity and their incapacity to leave the initiation
complex and permit elongation, we present a rationale for the dominant inhibitory effect of the two mutant IF2 proteins.
 |
EXPERIMENTAL PROCEDURES |
Strains and Media--
Strains and plasmids used are described
in Table I. All strains were grown in Luria Bertani (LB) medium. Where
indicated, ampicillin (Amp) was added at 100 µg/ml, chloramphenicol
(Cm) at 10 µg/ml, and spectinomycin at 100 µg/ml.
Plasmid Constructs--
Plasmids overexpressing infB
mutant alleles were constructed by exchanging a 1135-base pair
SnaB1-SstI DNA fragment in pSL4 (21) with the
corresponding fragments of pB18V400G and pB18H448E (4), giving rise to
pSL4V400G and pSL4H448E. In these plasmids, expression of the mutant
infB genes is under control of the thermoinducible pL and pR
promotors. The correct insertion and the presence of the mutation in
the inserted fragments were confirmed by DNA sequencing (22).
Construction of Strain SL679R--
In order to purify the
mutated IF2
proteins without contamination of the wild-type protein
we constructed a strain (SL679R) that expresses only the short form
(
) of the factor. The 1.6-kilobase BglII-HindIII fragment of pB18
45 (23),
carrying the mutant infB gene expressing only the
form
of IF2 was cloned into the low copy number plasmid pCL1920 (24)
digested with the same enzymes. The resulting plasmid, pCL
infB45,
was used to transform the strain SL598R (21). Curing of the
infB transducing phage was performed as described in Ref.
21. This gave rise to strain SL679R that expresses only the short form
of the factor.
Protein Purification--
Strain SL679R was transformed with the
plasmids pSL4V400G and pSL4H448E. The strains were grown at 30 °C in
LB medium in the presence of ampicillin and chloramphenicol to an
OD550 = 1, then the temperature was shifted to 42 °C to
allow expression of the mutant proteins from the thermoinducible pLpR
promoters and incubation was carried on for 2 more hours. The culture
was chilled on ice, the cells harvested, washed, and kept frozen at
80 °C. Overexpression of the mutant IF2 proteins was monitored by
10% SDS-polyacrylamide gel electrophoresis gels and immunoblotting of
aliquots at different times during heat induction.
Cell disruption and preparation of a crude extract was performed as
described (25). A 50% ammonium sulfate precipitate was resuspended in
LSB buffer (20 mM Hepes-KOH, pH 7.2, 1 mM
MgOAc, 7 mM
-mercaptoethanol, 5% glycerol) and dialyzed
against the same buffer for 2 × 1 h. Proteins were then
loaded on a Mono Q column (Pharmacia) and eluted with a 200-500
mM KCl gradient in LSB buffer. Fractions containing IF2
protein were pooled and, after lowering the salt concentration to about
100 mM by dilution with LSB buffer, loaded on a Mono S
column (Pharmacia) and separated by a 200-400 mM KCl
gradient in LSB buffer.
Preparation of Ribosomes--
Ribosome tight couples (70 S) and
ribosomal subunits (30 S and 50 S) were isolated from E. coli MRE600 cells, freshly harvested from exponential growth
phase, based on methods described in detail elsewhere (26) except that
the ribosomal subunits were fractionated on a 10-30% (w/w) hyperbolic
sucrose gradient in a Beckmann SW28 rotor at 27,000 rpm at 4 °C for
14 h.
Uncoupled GTPase Activity of IF2--
IF2-catalyzed
GTP hydrolysis was measured as described (10, 27). Reactions (25 µl)
were stopped with a mixture of 100 µl of 1 M perchloric
acid and 1 ml of 1 M KH2PO4 and the amount of [32P]phosphate liberated during the reaction
was determined as described previously (28). Kinetic constants were
determined from Lineweaver-Burk plots based on initial rate
measurements carried out with [
-32P]GTP concentrations
of 5-50 µM, which is in the Km range of wild-type IF2.
IF2-dependent Binding of
fMet-tRNAfMet to 70 S
Ribosomes--
Reactions contained limiting amounts of IF2 with
respect to 30 S and 50 S ribosomal subunits, initiation factors IF1 and
IF3, polyAUG and labeled
f[3H]Met-tRNAfMet. The
exact concentrations are given in the legend to Fig. 3. The reactions
were incubated at 25 °C for 5 min. Ribosome bound fMet-tRNAfMet was measured by filter
binding assay (29). The effect of GDP on the
fMet-tRNAfMet binding reaction was
studied by keeping the GTP concentration in the reaction constant at
0.1 mM and varying the GDP concentration in steps from 0 to
10 mM GDP (0.01, 0.1, 1, and 10 mM).
Sucrose Gradient Fractionation of the Puromycin
Reactions--
The fMet-tRNAfMet
binding reaction was carried out for 15 min and the puromycin reaction
for 20 min. The reaction buffer was the same as described for
fMet-tRNAfMet binding except that the
magnesium acetate concentration was 10 mM. The reactions
were diluted with 100 µl of cold reaction buffer, chilled on ice, and
loaded on a 7-20% sucrose gradient in the same buffer. The gradients
were centrifuged at 23,000 rpm in a Beckmann SW41 rotor for 12.5 h. 500-µl fractions were collected and proteins were precipitated
with trichloracetic acid and dissolved in SDS-polyacrylamide gel
electrophoresis sample buffer. The fractions were separated on 10%
polyacrylamide gels and analyzed by Western blotting with anti-IF2 antibodies.
 |
RESULTS |
Purification of Mutant Forms of IF2--
The E. coli
translation initiation factor IF2 exists as two forms in the cell,
IF2
and IF2
, which support bacterial growth equally well under
normal growth conditions (23). In order to purify the large form,
IF2
, of two IF2 mutants (V400G and H448E) without contamination by
wild-type protein we constructed a strain (SL679R, Table
I) expressing only the short form IF2
.
This strain is a derivative of SL598R (21) and carries the same
replacement of the chromosomal copy of the infB gene by a
cat cassette. Survival of the cell is assured by the
recombinant plasmid pCL
infB45 expressing solely the
form of the
factor. The mutated infB alleles are supplied by a second
compatible plasmid and their expression is controlled by the
thermoinducible promoters pRpL. Expression of the mutant proteins was
induced by shifting the temperature of the culture from 30 °C to
42 °C. The
-form of both mutant proteins was purified from these
strains by fast protein liquid chromatography techniques described
under "Experimental Procedures" and tested in a variety of in
vitro assays.
Uncoupled GTPase Activity--
As shown previously (4), both IF2
mutations, V400G and H448E, are lethal to E. coli. To
determine whether they were affected in their ability to hydrolyze GTP
in vitro, we performed an uncoupled GTPase assay, where the
initial rate of [
-32P]GTP hydrolysis by IF2 was
measured in the presence of 70 S ribosomes, but in the absence of any
tRNA or mRNA. As shown in Fig. 1,
both mutants have practically lost their ability to hydrolyze GTP.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Initial rate measurements of the uncoupled
GTPase activity of wild-type and two mutant forms (V400G and H448E) of
IF2. Reactions were carried out at 37 °C. A single incubation
mixture (25 µl) contained 50 mM ammonium chloride, 7 mM -mercaptoethanol, 17 pmol of each 30 S + 50 S
ribosomal subunits, 800 pmol of [ -32P]GTP (specific
activity: 660 cpm/pmol), 10 mM magnesium acetate, and 3 pmol of wild-type or mutant IF2 proteins and was scaled up according to
the number of samples taken during the time course of the reaction.
Samples of the reaction were taken at the indicated intervals and the
amount of [32P]phosphate liberated during the reaction
was measured as described previously (28). Values were corrected for
the volumes and background GTP hydrolysis detected in the absence of
factor. , wild-type IF2; , IF2 V400G; , IF2 H448E.
|
|
We also measured the initial rates of GTP hydrolysis by wild-type and
mutant forms of IF2 as a function of increasing GTP concentration. The
resulting double-reciprocal plot is shown in Fig.
2. Determination of the
Km values derived from Fig. 2 revealed that the
affinity of neither of the mutant proteins for GTP is reduced compared
with wild-type IF2 (Table II). On the
contrary, the respective Km values are slightly
lower than that of the wild-type protein, especially in the case of the
V400G mutant (15.3 µM versus 30 µM for the wild-type protein).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Lineweaver-Burk plots of
ribosome-dependent GTPase activity of wild-type and mutant
forms of IF2. Reactions were as described in the legend to Fig. 1
except that they contained 37 pmol of 70 S (30 S + 50 S), 19 pmol of
factor, and various concentrations of GTP (5-50 µM) and
were carried out for 10 min. Hydrolysis rate (V) is given in mole of
inorganic phosphate liberated per mole of IF2 1
min 1. , wild-type IF2; , IF2 V400G; , IF2
H448E.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Km and Vmax values for the
uncoupled GTPase activity of wild-type and mutant IF2 proteins.
Vmax is expressed as moles of GTP hydrolyzed per
mole of factor per minute.
|
|
The velocity of the uncoupled GTPase activity of the mutant proteins is
strongly reduced compared with that of the wild-type IF2, however. As
determined from Lineweaver-Burk plots (Fig. 2), the V400G and the H448E
mutants exhibit 11.4- and 40-fold lower Vmax
values than the wild-type protein, respectively (Table II). This
clearly indicates that it is the catalytic activity, and not the
ability to bind the substrate, that is impaired in the mutants. On the
other hand, this loss of activity is not sufficient in itself to
explain their dominant negative behavior over the wild-type protein
in vivo.
Binding of fMet-tRNAfMet to
the Ribosome--
The main role of IF2 in translation initiation is to
promote fMet-tRNAfMet binding to the
ribosome. We tested the ability of both mutant proteins to promote
fMet-tRNAfMet binding to 70 S ribosomes
(30 S + 50 S subunits) in the presence of translation initiation
factors IF1 and IF3. Fig. 3 shows the initial rate of factor-dependent
fMet-tRNAfMet binding for the different
IF2 proteins. The V400G mutant is 1.5-fold more active and the H448E
mutant 1.3-fold less active than the wild-type IF2. We also measured
the efficiency of fMet-tRNAfMet binding
as a function of GTP concentration. The capacity to promote this
reaction increases for all three proteins in the 0 to 1 mM GTP range (Fig. 4). In this assay the
activity of the V400G mutant is slightly lower than that observed with
wild-type IF2. The H448E mutant promotes about 60% of tRNA binding
compared with the wild-type protein (Fig. 4), but this level still
represents a full stoichiometric binding of
fMet-tRNAfMet.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Initial rate of
fMet-tRNAfMet binding to the
ribosome in the presence of wild-type and mutant forms of IF2. A
single incubation mixture (50 µl) contained 10 mM
Tris-HCl, pH 7.4, 100 mM NH4Cl, 3.5 mM magnesium acetate, 1 mM dithiothreitol, 1 mM GTP, 3.3 µg of poly(A,U,G), 28 pmol of each 30 S + 50 S ribosomal subunits, IF1 and IF3, 4 pmol of IF2 (wt or mutant), 22.5 pmol f-[3H]Met-tRNA (specific activity 2000 cpm/pmol).
Reactions were scaled up according to the number of samples to be taken
and incubated at 25 °C. Samples were taken at the indicated
intervals and the amount of ribosome bound
f[3H]Met-tRNAfMet was
quantified by filter binding assay (29). The background due to
f-[3H]Met-tRNAfMet binding
in the absence of wild-type or mutant IF2 was subtracted. ,
wild-type IF2; , IF2 V400G; , IF2 H448E.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of the GTP concentration on the
binding of fMet-tRNAfMet to the
ribosome promoted by wild-type and mutant IF2 proteins. Reactions
were set up as described in the legend to Fig. 3 except that the GTP
concentration was varied. The incubation time was 5 min at 25 °C.
, wild-type IF2; , IF2 V400G; , IF2 H448E.
|
|
As described above, the binding of GTP to the mutant IF2 proteins as
analyzed by Km measurements of the uncoupled GTPase
activity is not impaired. In order to detect potential differences in
the sensitivity of the mutant proteins to the presence of GDP we
measured the inhibitory effect of various GDP concentrations on
fMet-tRNAfMet binding in the presence of
a constant GTP concentration (0.1 mM). The results are
summarized in Table III. The two mutant
IF2s appear to be slightly more inhibited at very low GDP
concentrations, but the overall pattern of inhibition is practically
identical for all three proteins.
View this table:
[in this window]
[in a new window]
|
Table III
Inhibitory effect of GDP on IF2-dependent
fMet-tRNAfMet binding to ribosomes (GTP = 0.1 mM)
fMet-tRNAfMet bound in the absence of GDP is considered
as 100% binding.
|
|
Puromycin Reaction--
The correct positioning of
fMet-tRNAfMet is promoted by IF2 and IF3
and is essential for peptide bond formation during the first round of
elongation. We measured the capacity of the mutant proteins to promote
formation of the first peptide bond in the puromycin reaction, as a
function of the GTP concentration (Fig.
5). Upon addition of GTP, the wild-type
protein and the V400G mutant quickly reach their full activity with an
only slight increase in efficiency in the 0.25 to 1 mM GTP
range. In contrast, no formation of fMet-puromycin can be observed,
regardless of the GTP concentration, when the initiation complex was
formed with the H448E IF2 mutant (Fig. 5).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of GTP concentration on the formation
of fMet-puromycin in 70 S initiation complexes containing wild-type or
mutant IF2 proteins. Following the incubation at 25 °C for 5 min, puromycin was added to a final concentration of 1 mM
to the reactions of the fMet-tRNAfMet
binding assay shown in Fig. 4, and incubated for another 5 min.
Formation of fMet-puromycin was assayed as described (43). Values in
Figs. 4 and 5 are from the same experiment and are directly comparable.
The background value due to formation of
f[3H]Met-puromycin in the absence of IF2 was subtracted.
, wild-type IF2; , IF2 V400G; , IF2 H448E.
|
|
If one relates these data to the quantity of
fMet-tRNAfMet bound to ribosome it
becomes apparent that in the presence of the V400G mutant almost 100%
of the fMet-tRNAfMet is converted to
fMet-puromycin (compare Figs. 4 and 5). On the other hand, based on the
quantity of fMet-tRNAfMet initially
bound, more than twice as much
puromycin-fMet-tRNAfMet is formed in the
presence of wild-type IF2. This suggests that IF2 and hence ribosome
recycling is more efficient with wild-type factor.
Recycling of IF2--
GTP hydrolysis is believed to be essential
for the recycling of IF2. We thus tested to see whether the wild-type
and the mutants proteins were still associated with the ribosome after
the puromycin reaction. This was done by separating the ribosomes
contained in the reaction mixture on sucrose gradients and by analyzing the different fractions for the presence or absence of IF2 by Western
blot (Fig. 6). In the case of the
wild-type protein, practically all the IF2 had left the ribosome after
the puromycin reaction and was present in the top fractions of the
gradient, where unbound proteins are to be found. In contrast, both
mutant proteins stay bound to the ribosome and are found in the 30 S,
50 S, and 70 S regions of the gradient. We thus conclude that neither
of the mutant factors are efficiently recycled following formation of the first peptide bond.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
Binding of wild-type and mutant IF2 proteins
to ribosomes after engagement of the 70 S initiation complex in peptide
elongation. Puromycin reactions were carried out in the presence
of 1 mM GTP and immediately subjected to sucrose density
gradient centrifugation as described under "Experimental
Procedures." After gradient fractionation, the ribosomal profile
(top of the figure) was obtained by measuring the OD at 260 nm. Individual fractions were submitted to SDS-gel electrophoresis and
tested for the presence of IF2 by Western blot.
|
|
 |
DISCUSSION |
The role of GTP and its hydrolysis during translation initiation
has never been clear. Earlier in vitro studies, using
non-hydrolyzable GTP analogs, had suggested that GTP hydrolysis may be
important for the release of IF2 from the 70 S initiation complex
(30-33). We wished to carry out a mutational analysis to test this
hypothesis, to precisely define the amino acid residues involved in GTP
hydrolysis and to correlate the in vivo phenotype of a
mutant protein to its characteristics in vitro. If the
analogy between the EF-Tu G-domain structure and the three-dimensional
model for the IF2 G-domain holds true, then the IF2 V400G and H448E
mutants should only be impaired in their catalytic (GTPase) activity
but not in their affinity for the substrate as is the case for
equivalent (V20G) or similar (H84G) substitutions in EF-Tu (15, 19). This is exactly what we observed, the Vmax
values for the V400G and H448E mutants being 11.4- and 40-fold lower
than that observed for the wild-type GTPase, respectively. At the same
time, the affinity for GTP was not adversely affected, but rather
slightly increased, up to 2-fold in the case of the V400G mutant (Table II). It is interesting to note that the equivalent mutation in EF-Tu
(V20G) led, on the contrary, to a less efficient binding of GTP (15).
The V20G mutation in EF-Tu caused a 5-10-fold decrease in GTPase
activity and impaired its stimulation by the ribosome (14, 15). The
impaired GTPase activity of the IF2 V400G mutant thus indicates that
the capacity to hydrolyze GTP, although totally dependent on the
presence of the 50 S ribosomal subunit, resides within the IF2 protein itself.
The main function of IF2 in the cell is the binding and correct
positioning of fMet-tRNAfMet on the 70 S
ribosome for the subsequent elongation step. As shown in Fig. 3 both
wild-type and mutant proteins promote
fMet-tRNAfMet binding to the ribosome in
a GTP-dependent manner. In the absence of GTP, very little
or no binding was observed. Binding of
fMet-tRNAfMet to the ribosome is
especially sensitive to the presence of GTP when IF2 is present at low
(catalytic) concentrations, as is the case in the experiments reported
here (32-35). Both mutant proteins promote efficient
fMet-tRNAfMet binding to the ribosome
(Fig. 4). A comparison of initial rate measurements in the presence of
1 mM GTP (Fig. 3) revealed slight differences, with the
V400G mutant being 1.5 times faster than the wild-type protein. On the
other hand, the H448E mutant was somewhat slower (1.3-fold) than the
wild-type protein. At optimal GTP concentrations (1 mM),
the H448E mutant is still able to bind fMet-tRNAfMet in stoichiometric
quantities. The wild-type protein binds
fMet-tRNAfMet most efficiently, due to
factor recycling, but the V400G mutant also seems to exhibit some
catalytic activity (Fig. 4).
It was observed previously (6) that, at low concentrations of wild-type
IF2 and 30 S subunits, GDP acts as a competitive inhibitor of 30 S
initiation complex formation. We obtained comparable results when
measuring the formation of 70 S initiation complex in the presence of
limiting amounts of IF2. The capacity of both wild-type and mutant
proteins to promote fMet-tRNAfMet
binding to 70 S ribosomes was inhibited by GDP to roughly the same
extent in all three cases (Table III). This suggests that the overall
affinities for GDP and GTP are not significantly altered in either IF2
mutant relative to the wild-type protein.
Since the mutant IF2 proteins mediate
fMet-tRNAfMet binding to the ribosome,
this cannot account for their incapacity to support growth. However, a
difference between the wild-type and the mutant proteins was observed
in the formation of the first peptide bond, measured by the puromycin
reaction. Due to factor recycling, wild-type IF2 leads to the formation
of 2.5 times more fMet-puromycin than fMet-tRNAfMet initially bound (compare
Figs. 4 and 5). The H448E mutant is completely inactive in promoting
the formation of fMet-puromycin, the V400G mutant allows the
stoichiometric conversion of all bound fMet-tRNAfMet into fMet-puromycin,
indicating that it is not recycled. The activity of the IF2 V400G
mutant can be explained in two ways. Either the residual GTPase
activity is sufficient for some factor release to occur, or the
formation of fMet-puromycin can still take place even with the factor
bound to the ribosome. While we cannot rule out that some GTP
hydrolysis contributes to the overall activity observed, we rather
favor the second possibility. Formation of fMet-puromycin was assayed
under conditions close to those used for determining the initial rates
of the uncoupled GTPase (i.e. reaction time, IF2:ribosome
ratio) and where neither of the mutant proteins showed detectable
GTPase activity (Fig. 1). Moreover, puromycin is a very small molecule
compared with the EF-Tu·GTP·aa-tRNA ternary complex normally
involved in the formation of the first peptide bond and might still be
able to react with fMet-tRNAfMet
despite the presence of IF2. Experiments with a non-hydrolyzable GTP
analog (GDPCH2P) support this possibility. First
peptide bond formation in the presence of wild-type IF2 and
GDPCH2P still occurred substantially when assayed via the
formation of fMet-puromycin (36). Thus we believe that IF2 V400G does
not hydrolyze GTP and leave the ribosome but still permits near
stoichiometric conversion of bound
fMet-tRNAfMet into fMet-puromycin
(compare Figs. 4 and 5). In the case of the H448E mutant we speculate
that the fMet-tRNAfMet is not correctly
positioned with respect to the peptidyl-hydrolase center, or its
conformation is different to that of the wild-type or the V400G IF2,
such that the amino acid moiety on the tRNA is shielded, thereby
inhibiting the puromycin reaction. The differential behavior of the two
GTPase mutants also suggests that there is no direct link between GTP
hydrolysis and the first peptide bond formation. This conclusion is
supported by previous experiments where GTP was removed from the 30 S
initiation complex without altering the ability of the subsequent 70 S
complex to participate in peptide bond formation (33).
Studies in the 1970's, using non-hydrolyzable GTP analogues (30-33)
have led to the current belief that GTP hydrolysis is necessary for the
rapid release of IF2. Our experiments strongly support this view. When
the puromycin reactions are subjected to sucrose density gradient
centrifugation it is apparent that only the wild-type IF2 leaves the
ribosome. Several conclusions can be drawn from this observation. First
of all, the fact that the mutant factors are not released from the
ribosome after formation of fMet-puromycin provides a good explanation
for their dominant negative behavior in vivo. Translation
factor-dependent GTP hydrolysis involves an interaction
with the ribosomal GTPase center on the 50 S subunit which encompasses
the binding sites for L11 and the pentameric protein complex
L10·(L12)4 (37, 38). IF2 and EF-Tu have both been shown
to interact with this region of the ribosome (39-42). An IF2 molecule
which stays blocked on the ribosome would inhibit binding of the
ternary EF-Tu·GTP·aa-tRNA complex and consequently subsequent
elongation steps. The difference observed between the two mutants in
the puromycin reaction can only account for the correct positioning of
the fMet-tRNA on the ribosome, but not for its ability to promote
formation of the first peptide bond.
The question remains why the dominant negative effect of the IF2 V400G
mutant is stronger than that of the H448E mutant (4), despite a
slightly higher residual GTPase activity. A likely answer is provided
by the sucrose gradient experiment. After centrifugation, all of the
IF2 V400G cosediments with the different ribosomal particles and none
has dissociated sufficiently from the 70 S ribosome to be found at the
top of the gradient. On the other hand, the H448E mutant clearly
dissociates more easily as judged by a steady increase of the IF2
concentration toward the 30 S fractions and the top of the gradient. In
the case of the V400G mutant at least, the presence of factor in the 50 S region of the gradient reflects, to some extent, binding of IF2 to
this ribosomal subunit. Recentrifugation of the different gradient fractions through a sucrose cushion revealed some IF2 V400G associated with the 50 S subunit (data not shown). The increased ability of the
H448E mutant to dissociate from the ribosome, despite its practically
inexistent GTPase activity, probably explains why this mutant is less
inhibitory to bacterial growth in the presence of wild-type IF2. This
observation also underlines the importance of the conformational state
of IF2 for binding to the ribosome. Independent of the GTPase activity,
even slight alterations of the G-domain can have important consequences
for the rest of the protein, its affinity to the ribosome and the
positioning and reactivity of the
fMet-tRNAfMet. The weak, but inverse
effect on the affinity of IF2 for GTP, relative to the corresponding
V20G mutation in EF-Tu, highlights some structural differences between
the two factors. Nevertheless, our results essentially confirm the
close functional relationship between the EF-Tu and IF2 G-domains and
show the importance of the Val400 and His448
residues for GTP hydrolysis and IF2 recycling during formation of the
first peptide bond.