(Received for publication, June 29, 1995; and in revised form, September 6, 1995)
From the
The first step in the sequence of interactions between the
ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and
aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA,
is the codon-independent formation of an initial complex. We have
characterized the initial binding and the resulting complex by
time-resolved (stopped-flow) and steady-state fluorescence measurements
using several fluorescent tRNA derivatives. The complex is labile, with
rate constants of 6 10
M
s
and 24
s
(20 °C, 10 mM Mg
)
for binding and dissociation, respectively. Both thermodynamic and
activation parameters of initial binding were determined, and five
Mg
ions were estimated to participate in the
interaction. While a cognate ternary complex proceeds from initial
binding through codon recognition to rapid GTP hydrolysis, the rate
constant of GTP hydrolysis in the non-cognate complex is 4 orders of
magnitude lower, despite the rapid formation of the initial complex in
both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is
strongly affected by the presence of the tRNA. This suggests that
codon-anticodon recognition, which takes place after the formation of
the initial binding complex, provides a specific signal that triggers
fast GTP hydrolysis by EF-Tu on the ribosome.
Binding of aminoacyl-tRNA (aa-tRNA) ()to the
ribosomal A site takes place in a complex with elongation factor Tu
(EF-Tu) and GTP (``ternary complex'') and proceeds in several
steps. After the initial contact of the ternary complex with the
ribosome, codon recognition takes place, which is followed by the
hydrolysis of GTP, the dissociation of the factor, and the
accommodation of aa-tRNA in the A site. EF-Tu-binding sites were
localized at the base of the L7/L12 stalk on the 50 S subunit and on
the 30 S subunit in the upper part of the body(1, 2) .
Domain VI of 23 S RNA was identified to participate in the function of
EF-Tu(3, 4, 5) , and also the 530 loop of 16
S RNA seems to be in direct contact with EF-Tu(6) .
Additionally, a number of proteins of both large and small subunits
have been localized In the vicinity of A site-bound EF-Tu or
aminoacyl-tRNA.
We have shown recently that the first step in the
interaction between the ribosome and the EF-Tuaa-tRNA complex is
a codon-independent step, referred to as initial binding(7) .
After subsequent codon-anticodon recognition, a cognate ternary complex
is found in the pre-A site(8) , or recognition
site(9) , or T/A state(10) , in which the anticodon of
the aa-tRNA is bound to the mRNA, while the acceptor domain is still
bound to EF-Tu. In the absence of cognate codon recognition, the
EF-Tu
aa-tRNA complex does not enter this state and remains in the
initial binding state, from which it is rejected with high probability.
Initial binding and the initial complex have not been studied in detail
up to now.
In this paper, we characterize the initial binding and
the resulting complex with respect to kinetic and thermodynamic
characteristics. Our results show that the initial binding of
EF-TuGTP
aa-tRNA to the ribosome leads to a labile complex
with properties fitting to the initial screening of ternary complexes
by the ribosome. Most significantly, the rate constants of formation
and dissociation of the initial complex are such that they will not
limit appreciably the rate of cognate aa-tRNA binding under
physiologically relevant conditions. Furthermore, in the absence of
codon recognition, ribosome-mediated GTP hydrolysis in the
EF-Tu
GTP
aa-tRNA complex is extremely slow, in accordance
with a model where the GTPase of EF-Tu is triggered by cognate
codon-anticodon interaction(11) .
The complex eluted from the gel filtration column (1
µM) was stored on ice and diluted to the usual working
concentration (0.2 µM) immediately before the stopped-flow
experiment. The same procedure was applied to prepare the
EF-Tu
GTP complexes of Leu-tRNA
(Prf16,17,20) and
Phe-tRNA
(Flu8). The complexes containing
[
-
P]GTP were prepared in the same way,
except that nucleotide-free EF-Tu was used(18) .
The experiments
were performed by rapidly mixing equal volumes (60 µl each) of the
ternary complex, purified by gel filtration, and the ribosome complex
to give final concentrations of 0.1 and 0.3 µM,
respectively. If not stated otherwise, the temperature was 20 °C.
From experiment to experiment, the reproducibility of the rate
constants given is about ±10% and that of the amplitudes about
±15%; within one experiment, the reproducibility from shot to
shot was 5% for both parameters.
To determine rate constants of
initial binding, ribosome titrations were performed, i.e. rates were measured at a fixed concentration of
EF-TuGTP
aa-tRNA (0.1 µM), and increasing
concentrations of ribosome complex (from 0.3 up to 2.5
µM). To measure rates at ribosome concentrations lower
than 0.3 µM, experiments were performed at a 0.02
µM concentration of the ternary complex. Rate constants
were determined from the concentration dependence of the apparent rate
constants on the basis of a two-step model (19) ().
For states C and D, the relative fluorescence
quantum yields ( and
) were
calculated from the time course of the reaction at 20 °C and 10
mM Mg
, setting the initial fluorescence
(
) of the ternary complex to 1 and taking into account
the rate constants k
, k
, k
, k
determined from the titration. The
fitting was performed according to , which were derived
adopting previously described procedures (19) , (
)assuming pseudo first-order conditions with respect to the
ribosome
concentration:
where a and b
are the
initial concentrations of the ternary complex and ribosomes,
respectively; K
and K
are
binding constants; and and are equilibrium concentrations of A and D, respectively.
The dependence of the
fluorescence of A (free ternary complex) and of D (ribosome-bound ternary complex) on both Mg concentration and temperature was measured at a saturating
concentration of poly(A)-programed ribosomes. The fluorescence of
Phe-tRNA
(Prf16/17) both in the free ternary complex and
in the ribosome-bound state was not affected by Mg
(3.5-10 mM Mg
) (data not shown);
thus, the relative fluorescence quantum yields of states A, C, and D, measured at 10 mM
Mg
, were used for fitting the time courses of the
initial binding at lower Mg
concentrations for the
determination of the individual rate constants of the reaction. The
increase in temperature affected the fluorescence of both the free and
ribosome-bound ternary complexes. The respective temperature
coefficients, which were similar for both states, were taken into
account for the determination of the quantum yields of states A, C, and D for further fitting the rate
constants. The activation parameters of the initial binding reaction
were calculated from the temperature dependence of the rate
constants(19) .
To determine
the K of the initial binding complex, the
fluorescence of 0.1-0.2 µM purified
EF-Tu
GTP
[
C]Phe-tRNA
(Prf16/17)
was measured alone and with the addition of increasing amounts (up to
2.5 µM) of poly(A)-programed ribosomes with tRNA
in the P site. The resulting fluorescence was corrected for
dilution (<15%) and for background fluorescence (
2%). The data
were evaluated by fitting :
where F is the initial fluorescence of the
ternary complex; F
is the maximum fluorescence
after addition of saturating amounts of ribosomes; F(c) is the fluorescence at a given concentration of
free ribosomes, x; and c
and c
are the added concentrations of ribosomes and
ternary complex, respectively. The fitting program was TableCurve
(Jandel). The standard thermodynamic parameters of the interaction were
calculated from the temperature dependence of the K
. The number of Mg
ions
participating in the interaction was estimated as
described(21) .
Fluorescence quenching titrations were
performed by measuring the fluorescence intensity in the absence (F) and presence (F) of increasing
amounts of potassium iodide (up to 100 mM). To determine the
quenching constant, K
, the data were analyzed
according to the Stern-Volmer equation for collisional quenching as
described(22) .
where A and G denote cognate and non-cognate ternary complexes, respectively; B denotes ribosomes; and C, D, E, F, H, and I denote intermediates of the A site binding of cognate and non-cognate ternary complexes. The formation of the non-cognate initial complex, H, is followed by the slow formation of I (see ``Results''); this step is not observed in the cognate case(15) . Steps 2-4 represent codon-anticodon interaction, GTP hydrolysis, and peptide bond formation.
The
interaction of the ternary complex with poly(A)-misprogramed ribosomes
leads to a rapid increase in the fluorescence of proflavin in the
D-loop of either Phe-tRNA(Prf16/17) or
Leu-tRNA
(Prf16,17,20) (Fig. 1). With both tRNA
derivatives, the fluorescence increase is biphasic, with apparent rate
constants of about 40 and 3 s
, respectively. The
presence or absence of the mRNA does not influence the initial binding
since EF-Tu
GTP
Phe-tRNA
(Prf16/17) interacting
with vacant or with poly(A)-programed ribosomes exhibits the same
effects (Table 1). A fluorescence change is also observed with
fluorescein at position 8 (Table 1).
Figure 1:
Time course of initial binding of EF-Tu
GTP
1Phe-tRNA
(Prf16/17) (trace 1),
EF-Tu
GTP
Leu-tRNA
(Prf16,17,20) (trace
2), and EF-Tu
mant-dGTP
Phe-tRNA
(trace 3) to 70 S ribosomes programed with poly(A) and
carrying tRNA
in the P site and control without ribosomes (trace 4). Stopped-flow experiments were performed as
described under ``Materials and Methods.'' Parameters of the
two-exponential fits are as follows: 1) k
= 43 s
and A
= 6%, k
= 3 s
and A
= 2%; 2) k
= 41 s
and A
= 2%, k
= 3 s
and A
=
4%.
Neither wybutin in the
anticodon loop of tRNA nor mant-dGTP replacing GTP in the ternary
complex shows any significant fluorescence change upon binding of the
respective ternary complex of Phe-tRNA to
poly(A)-programed ribosomes ( Fig. 1and Table 1), in
contrast to the extensive signal changes observed with both labels upon
binding to ribosomes programed with poly(U)(11, 15) .
Replacement of wybutin at position 37 with proflavin also did not
result in the appearance of any fluorescence change (Table 1).
In analogous stopped-flow experiments with EF-TuGTP
Phe-tRNA
(Prf16/17) and ribosomal subunits, a slight
fluorescence increase with k
around 15
s
is detectable (<1%) (Table 1). This shows
that, although there seems to be an interaction of the ternary complex
with either subunit alone, the proper formation of the initial complex
requires 70 S ribosomes. The biochemical analysis shows that, upon
rapid filtration without prior dilution, a small and variable fraction
(10-15%) of the non-cognate complex is retained on Millipore
filters; however, the complex is quite unstable and readily dissociates
upon dilution shortly before filtration.
The accessibility of the
dye in Phe-tRNA(Prf16/17) in the initial complex to
solvent access was measured under equilibrium conditions upon adding KI
as a quencher. The interaction with poly(A)-programed ribosomes does
not change the quenching constant (K
= 7.5 M
), indicating that there is no direct
contact of the ribosome with the dye, but rather that the fluorescence
change is due to a conformational change around the fluorophore. This
conclusion is supported by preliminary results of fluorescence
lifetimes measurements that show that, upon initial binding, the three
lifetimes remain the same, but their relative contribution is changed. (
)
Figure 2:
Effect
of preoccupancy of the A site with AcPhe-tRNA on the
binding of EF-Tu
GTP
Phe-tRNA
(Prf16/17).
The relative amount of Ac[
C]Phe-tRNA
bound (added) per ribosome was as follows: trace 1, 0.93
(1.1), P site blocked, A site free; trace 2, 1.32 (1.5), P
site blocked, A site partially blocked (
40%); trace 3,
1.97 (5.0), both P and A sites fully blocked; trace 4, control
without ribosomes. The amount of ribosome-bound
Ac[
C]Phe-tRNA
was the same before
and after binding the ternary complex. Parameters of the
two-exponential fit are as follows: k
=
9 s
and A
= 7%, k
= 2 s
and A
= 3%.
Figure 3:
Concentration dependence of k (A) and k
(B) of the binding of EF-Tu
GTP
Phe-tRNA
(Prf16/17) (
and
) or EF-Tu
GTP
Leu-tRNA
(Prf16,17,20) (
and
&cjs0822;
) to poly(A)-programed ribosomes. The lines represent the fits
according to the equations for
and
(see ``Material and Methods'') with the following
parameters: k
= 5.6
10
M
s
, k
= 24 s
, k
= 4 s
, and k
= 1.5
s
.
The
second step is characterized by the rate constants k = 4 ± 1 s
and k
= 1.5 ± 0.5
s
. The origin of this step is not clear at present.
It was observed with several preparations of ribosomes that all were
highly active. Also, neither k
nor k
was affected by the addition of
EF-Tu
GTP up to 5 µM (data not shown), thus excluding
any influence of a reaction involving free EF-Tu
GTP, such as the
rearrangement from the quinternary to the ternary complex (see
``Materials and Methods''). The relative amplitude of the
second step is larger for Leu-tRNA
(Prf16,17,20) than for
Phe-tRNA
(Prf16/17). This may indicate that the slow step
reflects a local movement of the fluorophore that is particular for
each labeled tRNAs species and is not of immediate relevance for the
mechanism of A site binding.
Figure 4:
Mg dependence of the
affinity of the initial complex of EF-Tu
GTP
Phe-tRNA
(Prf16/17) with poly(A)-programed ribosomes. A, titration of EF-Tu
GTP
Phe-tRNA
(Prf16/17) with increasing concentrations of
ribosomes at 10 mM (
), 7.5 mM (
), 6.5
mM (
), and 5 mM (
&cjs0822;)
MgCl
; B, determination of the number of
Mg
ions participating in the formation of the initial
complex. K
values were determined from
titrations, examples of which are shown in A.
The
affinity of the ternary complex for misprogramed ribosomes increases by
>100-fold when the Mg concentration is increased
from 5 to 12.5 mM (Fig. 4). The slope of the linear
plot of log(K
) versus log(Mg
) (Fig. 4B) reveals that
five Mg
ions are involved in initial
binding(21) . Extrapolation to a physiologically relevant
Mg
concentration (<3 mM) yields a K
of the initial binding complex of 30
µM. At the Mg
concentration used in the
stopped-flow experiments (10 mM), the titration gives K
= 0.15 µM, in agreement with
the value calculated from the rate constants (0.16 µM).
To estimate the thermodynamic parameters of the initial complex (Table 2), the K values obtained at
different temperatures were plotted according to van't
Hoff's equation. Extrapolation of the linear plots to 37 °C
gives K
= 0.24 µM at 10 mM Mg
. The negative values of the standard (298.15
K) enthalpy change (
H
) and the positive
values of the standard entropy change (
S
) (Table 2) indicate that the initial complex forms spontaneously
at any temperature.
Figure 5:
Temperature dependence of the rate
constants of initial binding of EF-Tu GTP
Phe-tRNA
(Prf16/17) to poly(A)-programed ribosomes.
, k
;
, k
;
, k
;
&cjs0822;, k
.
A decrease in the
Mg concentration strongly affects the initial binding
and results in a dramatic decrease in the amplitudes of both steps,
while the values of k
and k
remain constant (data not shown). Analysis of
the time course of the reaction at different Mg
concentrations (see ``Materials and Methods'') yields
the Mg
dependence of all four individual rate
constants (Fig. 6). The forward rate constant of the first step (k
) is affected most. Extrapolation to 37 °C
and 3.5 mM Mg
yields forward and backward
rate constants for the second-order step of k
= 1.2
10
M
s
and k
= 65
s
, respectively.
Figure 6:
Mg dependence of the
rate constants of initial binding of EF-Tu
GTP
Phe-tRNA
(Prf16/17) to poly(A)-programed ribosomes
containing tRNA
in the P site.
, k
;
, k
;
, k
;
&cjs0822;, k
.
The results of the modeling are shown in Fig. 7. In the presence of an excess of non-cognate ternary
complexes, the rate of cognate amino acid incorporation into the
polypeptide is decreased 5 times in the presence of a 240-fold
excess of non-cognate complexes, independent of the concentration of
the cognate complex. For the situation in vivo, this excess is
probably an overestimation (25) . Thus, the modeling suggests
that the rate of amino acid incorporation is only moderately affected
by the presence of non-cognate ternary complexes at physiological
concentrations.
Figure 7:
Modeling of the effect of non-cognate
ternary complexes on cognate amino acid incorporation. The
concentrations used for the modeling were 0.25 mM ribosomes
and 0.01 () or 0.1 mM (
) cognate ternary complex.
The time of half-completion of the peptidyl transfer reaction
(
) was calculated (see ``Materials and
Methods''), divided by the value in the absence of competitor, and
plotted against the concentration of non-cognate
complex.
Figure 8:
GTP
hydrolysis in EF-Tu [
-
P]GTP
Phe-tRNA
on misprogramed ribosomes. A, k
in the presence of 0.3
µM (
), 1 µM (
), 1.5
µM (
&cjs0605;), or 2.5 µM (
)
poly(A)-programed, P site-blocked ribosomes; B, dependence of k
on the ribosome concentration.
The value at saturation equals the first-order rate constant of GTP
hydrolysis, k
= 1.7
10
s
.
In the ternary complex, the conformation of aa-tRNA is slightly different from that in solution; the main differences were found in the association of the T- and D-loops and in the core region(26, 27, 28, 29, 30, 31, 32) . Our data suggest that, upon initial interaction of the ternary complex with the ribosome, this region of the tRNA molecule is affected and changes the conformation. Subsequently, codon-anticodon recognition leads to yet another, and probably more extensive, rearrangement of this region of the tRNA. The latter rearrangement probably involves a transient unfolding of the D-loop that depends on cognate codon-anticodon interaction and hence may have an important role in the signal transduction from the site of codon-anticodon interaction to EF-Tu(11, 15) .
At
physiological concentrations of ternary complexes and ribosomes, the
rate of the initial binding of the cognate ternary complex is not
rate-limiting for A site binding anyway since the reaction is running
at a rate of 300 s
(k
= 1.2
10
M
s
, ribosome concentration of 250 µM).
It is to be noted that these conclusions may be restricted to
conditions of exponential growth, i.e. high concentrations of
ribosomes and ternary complexes. It is conceivable that a decrease in
the concentrations of ribosomes and/or ternary complexes, e.g. in a situation of limited supply, may create a situation in the
cell where the rate of initial binding may become rate-limiting for
elongation.
Our data
clearly show that the ribosome interactions of the non-cognate ternary
complex and the binary EF-TuGTP complex are grossly different, as
evident from the comparison of the rate constants of binding and
dissociation of the non-cognate ternary complex (6
10
M
s
and
24
, respectively) and of the binary complex (5
10
M
s
and 10
s
, respectively)
determined under comparable conditions(37) . The most important
difference, however, comes from the measurements of the rate constants
of GTP hydrolysis for the two types of complexes. For the binary
complex, the rate of GTP hydrolysis is limited by the low rate of
binding of EF-Tu
GTP to the ribosome. In contrast, for the ternary
complex (cognate or non-cognate), the rate of the bimolecular reaction
(6
10
M
s
) is not limiting, at least not at the
concentrations we have used, and nevertheless, the rate constant of
ribosome-induced GTPase in the non-cognate ternary complex is 2
10
s
, only 40 times faster than
the rate constant of the intrinsic GTPase in the unbound complex (5
10
s
). In comparison, in
the presence of correctly programed ribosomes, the rate of GTP
hydrolysis is 12 s
(11) , 4 orders of
magnitude faster than the rate of intrinsic GTP hydrolysis. Thus, in
the non-cognate situation, GTP hydrolysis does not occur in the
physiologically relevant time range and therefore cannot be implied as
a kinetic standard in the tRNA-independent rejection of
EF-Tu
GTP
aa-tRNA complexes.