The ribosome is the missing guanine-nucleotide exchange factor for EF-G
It has been reported that EF-G from Escherichia coli
binds to GTP with ten-fold lower affinity than it binds to GDP [12
]. On the assumption that there is a ten-fold excess of GTP over GDP in the cytoplasm and rapid nucleotide exchange on free EF-G, it was suggested that the rate-limiting step of guanine-nucleotide exchange in the EF-G cycle is the dissociation of EF-G•GDP from the postT ribosome [3
Our earlier data, showing the ribosome to be a GEF for RF3 [9
], prompted us to re-check the binding of free EF-G to GDP or GTP. The dissociation constant (KD
) for the EF-G•GDP complex was about 9 μM (Figure ), close to an earlier estimate of 4 μM [12
]. Results from experiments in which [3
H]-GDP in complex with EF-G was chased with unlabeled and further purified GTP [9
] (see below and Figure 4a for purification details), however, show a 60-fold larger effective KD
-value for the binding of EF-G to GTP than to GDP (Figure ). This factor of 60 provides a lower boundary to the correct value, because purified GTP solutions do contain some fraction of GDP from the hydrolysis of GTP. The intracellular GTP:GDP ratio has been estimated as 7:1 for Salmonella enterica
serovar Typhimurium [13
], and is probably similar in E. coli.
This suggests that a major fraction of free EF-G in E. coli
is bound to GDP.
Figure 2 Ribosome-dependent exchange of GDP to GTP on EF-G. (a) Scatchard plot from a nitrocellulose-filtration experiment to obtain the dissociation constant for the binding of [3H]-GDP to free EF-G. (b) Chase of [3H]-GDP from free EF-G by unlabeled GTP or, as (more ...)
If binding of EF-G to the pre-translocation (preT) ribosome required the factor to be bound to GTP, this would significantly reduce the rate of association of EF-G with the ribosome. This problem would, however, be eliminated if EF-G in the GDP-bound form associated rapidly with the ribosome and GDP-to-GTP exchange took place on, rather than off, the ribosome. To test the latter two hypotheses, we prepared preT ribosomes with fMet-Ile-tRNAIle and its corresponding codon in the A site and a UAA stop codon immediately downstream from the Ile codon. Translocation was catalyzed by EF-G at such a small concentration that each EF-G molecule had to cycle many times to obtain a significant fraction of translocated ribosomes. The concentration of GTP was fixed at 0.5 mM during incubations with varying concentrations of GDP, and the ribosome concentration chosen was sufficiently low that the rate of translocation per ribosome was approximated by the concentration of free EF-G multiplied by its effective association rate constant (kcat/Km) for ribosome binding (see Materials and methods). Because translocation brought the stop codon UAA into the ribosomal A site, the extent of translocation was conveniently quantified as the fraction of fMet-Ile peptide that could be rapidly released by RF2, when RF2 was added to a concentration in excess of that of the ribosomes at varying incubation times (Figure ).
We obtained 50% inhibition of the rate of EF-G recycling at 0.25 mM GDP, at which concentration the concentration of EF-G•GDP (KD = 9 μM) must have been at least 30 times larger than the concentration of EF-G•GTP (KD > 0.6 mM). If entry of EF-G to the ribosome had required the EF-G•GTP complex, this would have led to a 30-fold, rather than the observed two-fold, inhibition of translocation at 0.25 mM GDP (see Materials and methods). This implies that EF-G must have entered the ribosome in complex with GDP, and that the exchange of GDP for GTP must have taken place on, rather than off, the ribosome. The parameters that determine how the kcat/Km value for the entry of EF-G to the preT ribosome complex depends on varying ratios of GDP to GTP are defined in Materials and methods for a particular kinetic scheme.
The preT ribosome contains a deacylated tRNA in the P site (Figure ), which may be important for the GDP-to-GTP exchange reaction. This is suggested by experiments on guanine-nucleotide binding to EF-G in another type of ribosome complex. Here, EF-G was incubated with [3H]-GDPNP and either post-termination (postTerm) or naked ribosomes at varying concentrations of unlabeled GDP (Figure ). The postTerm ribosome has a deacylated tRNA in the P site and an empty A site programmed with a stop codon (Figure ), while the naked ribosome lacks ligands. The fraction of [3H]-GDPNP retained on a nitrocellulose filter, corresponding to ribosome-bound EF-G• [3H]-GDPNP, was reduced to 50% at a 160-fold excess of GDP in the postTerm case, or a 13-fold excess for the naked ribosomes. This implies that EF-G, bound to either type of ribosome, had much higher affinity for GDPNP than for GDP, and that the difference was more pronounced for postTerm than for naked ribosomes (Figure ). Accordingly, the presence of a deacylated tRNA in the P site of the preT ribosome led to more stable binding of EF-G• [3H]-GDPNP to this complex than to the naked ribosome. A corresponding stabilization of the EF-G•GTP complex on preT ribosomes by the P-site tRNA is expected, and would contribute to efficient guanine-nucleotide exchange (Figure ).
So far, we have not addressed the question of whether formation of a complex between EF-G•GDP and the preT ribosome leads directly to guanosine exchange, or whether the exchange reaction is preceded by a change in conformation of the ribosome. This problem is addressed in the next section.
EF-G•GDP drives the preT ribosome into a state that has hybrid tRNA sites
EF-G•GTP binds poorly to the pre-termination (preTerm) ribosome with a peptidyl-tRNA in the P site and an empty A site programmed with a stop codon (Figure ), but binds with high affinity to the postTerm ribosome with a deacylated tRNA in the P site [8
] (Figure ). In the latter case, cryo-EM results show the postTerm ribosome in a ratcheted state with the P-site tRNA in the hybrid P/E site [5
]. This suggests that high-affinity binding of EF-G•GTP to the ribosome requires the ratcheted state with hybrid tRNA sites; this state cannot be formed when there is peptidyl-tRNA in the P site. It is likely that the ratcheted ribosome conformation appears also in the translocation process, suggesting that EF-G•GDP can move the preT ribosome from the relaxed state, with three full binding sites for the tRNAs [5
], to the ratcheted state, with no E site binding and only two binding sites for tRNA [14
]. This would facilitate rapid GDP-to-GTP exchange on EF-G, and we have tested one of the predictions that emerges from this hypothesis, namely that the apparent affinity of a deacylated tRNA for the E site of the preT ribosome will be reduced by the addition of EF-G•GDP. This prediction was confirmed by an experiment showing that the affinity of tRNAfMet
for the E site of the preT ribosome was successively reduced by increasing amounts of EF-G in the presence of GDP (Table , set 1).
Dissociation constants for the binding of tRNAfMet and tRNAPhe to different ribosomal complexes
In order to monitor the translocation events that follow guanine-exchange on EF-G on the preT ribosome, we used A-site-specific cleavage of the mRNA by the bacterial toxin RelE, and this is described next.
Translocation events monitored by RelE cleavage of the A-site codon
RelE cuts mRNA specifically within the ribosomal A site [11
], and we used this activity to monitor ribosome movement along mRNA in the translocation steps (Figure ). An initiation complex (Init; Figure ) with fMet-tRNAfMet
in the P site was constituted by incubating ribosomes in the presence of initiation factors IF1, IF2 and IF3, fMet-tRNAfMet
, and 33
P-end-labeled mRNA encoding the dipeptide Met-Ile-stop (AUG AUU UAA). Exposure of this complex to RelE led to unique cleavage of the A-site codon to AU*U (Figure , lane 2). The Init complex (Figure ) was then converted to the preT complex (Figure ) by addition of the ternary EF-Tu•GTP•Ile-tRNAIle
complex. The resulting presence of fMet-Ile-tRNAIle
in the A site blocked the entry of RelE to the A site and reduced the rate of cleavage of the AUU codon (Figure , lane 3). Addition of EF-G•GTP to the preT complex catalyzed rapid translocation of fMet-Ile-tRNAIle
from the A to the P site, generating the postT complex (Figure ), and moved the stop codon into the A site of the postT complex, where it was rapidly cleaved by RelE (Figure , line 4).
Figure 3 RelE cleavage of mRNA in the A site of ribosomal complexes. (a) The mRNA fragments resulting from RelE cleavage in the A site of the three ribosomal complexes Init (see Figure 1a), preT (see Figure 1b) and postT (see Figure 1c), separated on a 10% sequencing (more ...)
Complete translocation requires GTP and GTP hydrolysis
In order to study further the guanine-nucleotide dependence of the translocation steps, the ribosomal preT complex was first separated from all other components of the translation mixture [8
]. RelE cleavage of the A-site codon was monitored after addition of EF-G to the purified preT complex in the presence of GTP, GDP or the non-cleavable GTP analog GDPNP (Figure ). In one type of experiment, the preT complex was first incubated with EF-G and either GTP or GDP for 10, 25 or 40 min and then the ribosomes were exposed to RelE for 5 min. In the presence of GTP, there was extensive cleavage by RelE of the stop codon (Figure , + GTP), meaning that a major fraction of the ribosomes had moved from the preT to the postT state.
In the presence of GDP, there was no significant RelE-dependent cleavage of the stop codon in the A site, even during the longest incubation time of 45 minutes (Figure , + GDP), meaning that the ribosomes had remained in their preT state during the whole incubation period. This implies that EF-G and GDP were unable to promote translocation, in apparent contradiction to previous results, showing rapid translocation by EF-G and GDP [7
]. We have noted that GTP contamination, common in commercial preparations of GDP, can have profound effects on the GTPases of protein synthesis. A typical elution profile (Figure ) shows such a GDP preparation to contain between 1 and 2% GTP, and the effect of this low level of contamination was studied in an experiment in which translocation of fMet-[14
C]-Ile-tRNA from the A site to the P site was probed by the fraction of peptide that could be rapidly released by RF2. The rate of translocation was insignificant with purified GDP, intermediate with unpurified GDP or with purified GDP + 2% GTP and fast with GTP (Figure ). Similarly, no translocation with pure GDP was detected by assessing the RelE-dependent cleavage of the mRNA (Figure ). Our nucleotide preparations were further purified by ion exchange chromatography on a MonoQ column [9
], while those of Rodnina et al.
] were not. This suggests that their 'GDP-dependent translocation' was, in fact, due to contaminating GTP. At such a large excess of GDP, the guanine-exchange reaction on the preT ribosome is expected to be the rate-limiting step for translocation, and this will lead to slow, monophasic translocation, exactly as they observed (see Materials and methods) [7
Figure 4 Contamination of GDP preparations with GTP strongly stimulates translocation by EF-G. (a) Elution profile of commercially available GDP from a MonoQ column showing the GTP and GMP contaminations. %B is the percentage of buffer B (20 mM Tris-HCl, 1 M NaCl) (more ...)
In the presence of GDPNP, about 11% of the stop codons were cleaved after addition of RelE, irrespective of the time of exposure of preT ribosomes to EF-G and GDPNP (Figure , + GDPNP 2). In a similar experiment, modified so that RelE was present from the start of the incubation of preT ribosomes with EF-G and GDPNP, the fraction of cleaved stop codons increased slowly with time (Figure , + GDPNP 1). This means that EF-G and GDPNP drove the ribosomes to a state that remained stable during the 45 min incubation in the absence of RelE (Figure , + GDPNP 1). In this state, the stop codon was partially available for RelE-mediated cleavage in the A site, resulting in very slow truncation of the mRNA (Figure , + GDPNP 2). A priori, this ribosomal state could be the postT state of the ribosome or a novel transition state ('transT*') in the translocation process where, in both cases, RelE-mediated cleavage of the stop codon in the A site was inhibited by ribosome-bound EF-G•GDPNP. An experiment in which the rates of RelE cleavage in the A-site codons of ribosomes in the putatively new state and postT ribosomes were compared at the same concentrations of EF-G and GDPNP (Figure ) showed that RelE cleaved the mRNA in the postT complex much faster than the mRNA on the ribosomes in the unknown state complex, proving that the ribosomal complexes could not have been the same. This means that the unknown state was transT*, and in the next section we characterize these complexes with respect to tRNA-exchangeability.
Exchangeability of tRNAfMet in preT, transT* and postT ribosomes
We characterized the transT* state with respect to the exchangeability of its deacylated tRNAfMet. First, we used nitrocellulose filtration to study dissociation of [33P]-tRNAfMet, originally in the P site of the preT complex (Figure ), from ribosomes incubated with EF-G together with GDP, GTP or GDPNP. In one type of experiment, the fraction of ribosome-bound [33P]-tRNAfMet was monitored as a function of time in the presence of either unlabeled tRNAfMet or tRNAPhe at fixed concentrations (Figure ). In another type of experiment, the fraction of ribosome-bound [33P]-tRNAfMet was monitored at a fixed time while varying the concentrations of unlabeled tRNAfMet or tRNAPhe (Figure ).
Figure 5 Properties of the transition state. (a) Time-dependent exchange of [33P]-tRNAfMet bound to the P site of 70 nM preT complex with 1 μM unlabeled tRNAfMet or tRNAPhe after the addition of 2 μM EF-G and 1 mM nucleotide. (b) The fraction of (more ...)
In the GDP experiment in which no translocation occurred (Figure , + GDP), there was no significant removal of [33
from the ribosome during 6 min in the presence of any unlabeled tRNA, as would be expected for ribosomes with deacylated tRNAfMet
stably bound to the P site after peptidyl transfer (Figure ). In the GTP case, in which there was rapid translocation (Figure , + GTP), there was fast dissociation of [33
in the presence of either tRNAfMet
(Figure ). The titration experiment (Figure ) shows that one fraction of [33
dissociated from the postT ribosomes in the absence of chasing tRNAs, and that the remaining fraction could be titrated out with either tRNAfMet
. These results reflect the comparatively low affinity of [33
for the E site and the lack of codon specificity for the E-site-bound tRNAs ([15
]; see also below).
In the case of GDPNP, [33P]-tRNAfMet dissociated slowly in the presence of tRNAfMet, but there was no dissociation in the presence of tRNAPhe, suggesting high affinity for [33P]-tRNAfMet and retained codon-specificity for deacylated tRNAs (Figure ). In line with this, the titration experiment (Figure ) shows that [33P]-tRNAfMet could be exchanged with unlabeled tRNAfMet but not with unlabeled tRNAPhe.
In a third type of experiment, [33
was chased with unlabeled tRNAfMet
from preT ribosomes incubated for a fixed amount of time in the presence of EF-G at a constant concentration and GDPNP at varying concentrations (Figure ). The fraction of ribosomes lacking [33
increased from 0 to 50% when GDPNP was varied from 0 to 40 μM and increased further to almost 100% at 250 μM GDP. This result shows that the affinity of EF-G•GDPNP for the transT* ribosome, containing one deacylated and one peptidyl tRNA, was approximately 100 times weaker than the affinity of EF-G•GDPNP for the postTerm ribosome, containing only one deacylated tRNA (see below)[8
Another experiment (Figure ) shows that tRNAPhe could not replace [33P]-tRNAfMet in transT* ribosomes, either with intact mRNA or with mRNA that had been cleaved by RelE. This means that the transT* ribosomes did not move to the postT state as a result of the mRNA cleavage, since that would have resulted in weak, non-selective E-site binding of the deacylated tRNAs (as shown in Table and Figure 7).
Addition of GDP to transT* ribosomes brings them back to the preT state
When GDP was added to transT* ribosomes, on which we have observed RelE-mediated cleavage of the stop codon to UA*A (Figure , + GDPNP 1; and Figure , + GDPNP), stop codon cleavage was completely eliminated and replaced by cleavage of the AUU codon (Figure , + GDPNP + GDP). The latter cleavage reaction was typical for the preT ribosome and occurred when the peptidyl-tRNA dissociated from the A site (Figure ). When, in contrast, GDP was added to postT ribosomes that were incubated in the presence of EF-G and GDPNP, the ribosomes remained in the postT state and there was rapid cleavage of the UAA codon (data not shown). These results strongly suggest that addition of GDP to the transT* ribosome brought it back to the preT state, providing further evidence that the transT* state is different from the postT state of the ribosome.
Figure 6 Removal of EF-G•GDPNP from the transition state with GDP. (a) Time-dependent cleavage of mRNA by 166 nM RelE in transT* complex in the presence of 2 μM EF-G and 0.32 mM GDPNP (GDPNP case) or after further addition of GDP to a concentration (more ...)
In line with previous results [8
], addition of EF-G•GDPNP to preT ribosomes brought them to a puromycin-reactive state (Figure ); puromycin mimics an aminoacyl tRNA and removes a nascent peptide from the ribosome by acting as a receptor in peptidyl-transfer. When GDP was also included, however, the puromycin-reactivity of the ribosomes was lost (Figure ), again showing that the resulting state could not have been the postT ribosome, which is fully reactive to puromycin [9
A deacylated [33P]-tRNAfMet in the transT* ribosome could readily be chased with unlabeled tRNAfMet, but its exchange rate in the preT ribosome was almost zero (Figure ). If GDP addition brought the transT* ribosome back to the preT state, one would therefore expect the exchange rate of the tRNAfMet to drop drastically. This prediction was nicely confirmed by experiments showing that addition of GDP to transT* ribosomes did indeed prevent exchange of [33P]-tRNAfMet with tRNAfMet (Figure ).
When release factor RF2 was added to transT* ribosomes, there was slow release of peptide (Figure ), suggesting that there was partial availability of the UAA stop codon in the A site, a necessary condition for termination by class-1 release factors [8
]. Addition of GDP to transT* ribosomes made them non-reactive not only to puromycin (Figure ), but also to peptide release induction by RF2 (Figure ). These mRNA cleavage results (Figure ), along with those for puromycin (Figure ), RF2 (Figure ) and tRNA exchange (Figure ) show that removal of EF-G•GDPNP from the transT* ribosome by the addition of GDP brought the ribosome back to the preT state with peptidyl-tRNA in the A site. This confirms that the transT* state cannot be identical to the postT state of the ribosome, and corroborates that transT* is a transition state in the translocation process, in which rapid hydrolysis of native GTP on EF-G normally occurs. When EF-G dissociated from the transT* ribosome, the mRNA rapidly slipped back to its preT position, but there was a short time during which RelE could cleave and RF1 could interact with the stop codon exposed in an EF-G-free A site.
Deacylated tRNAs bind to the ribosomal E site with low codon specificity
We showed above that [33P]-tRNAfMet could be chased by tRNAfMet but not by tRNAPhe in transT* (Figure ). This contrasts with E-site binding of deacylated tRNA, as follows. We designed experiments to obtain dissociation constants for the binding of deacylated tRNAfMet or tRNAPhe to the E site of postT ribosomes, programmed with Met (AUG), Phe (UUU) or Thr (ACG) codons. The binding of [33P]-tRNAfMet to the E site was assayed by nitrocellulose filtration, and a representative experiment with the Thr (ACG) codon in the E site is shown in Figure . Dissociation constants for the binding of tRNAPhe or tRNAThr to the differently programmed E sites of postT ribosomes were obtained as I50 values in competition experiments with a constant and almost saturating concentration of [33P]-tRNAfMet and varying concentrations of unlabeled tRNAPhe or tRNAThr (Figure ). The outcome of typical experiments, probing the binding of tRNAPhe to E sites programmed with Met, Phe or Thr codons, is shown in Figure , and all data are collected in Table . The results show that tRNAfMet and tRNAPhe bound to postT ribosomes with similar affinities and weak codon specificity. In similar experiments, we also found that the affinity of tRNAfMet for the E site was similar for postT ribosomes, postT ribosomes with RelE-mediated cleavage of the mRNA in the A site, and preT ribosomes, all with the same codon in the E site (Table ).
Figure 7 Binding of deacylated tRNA to the E site. (a) Binding of [33P]-tRNAfMet to the postT complex with fMet-Phe-Ile-tRNAIlein the P site and a Thr codon (ACG) in the E site. Insert: Scatchard plot to obtain the dissociation constant. (b) Chase of [33P]-tRNA (more ...)