Although we have not analyzed the kinetics quantitatively, the experiment clearly shows, in accordance with the chase experiment presented above, that mantGTP binds to RF3 on a PreTC, and the binding kinetics indicates that GTP may bind faster than GDP.
Thus, our measurements do not confirm the previous suggestion that the binding of GTP to ribosome-bound RF3 was precluded in the pre-termination state of the ribosome, whereas GDP dissociated and bound rapidly, independent of the functional state of the ribosome 17 , Previously, the affinities of guanine nucleotide binding to RF3 were determined by nitrocellulose filtration 17 , 18 , a non-equilibrium technique that is problematic for the quantification of kinetically unstable factor-nucleotide complexes The evaluation of the titration curves had to take into account the presence of close to one GDP molecule in RF3.
Thus, relative rather than absolute affinities were determined. The titration data were re-plotted to estimate the ratio between the K d values for the respective mant-labeled nucleotide and unmodified GDP Materials and Methods.
Assuming a K d value for GDP of 5 nM, as obtained by nitrocellulose filtration 17 , which is probably valid given the reasonable kinetic stability of the complex, the affinity of mantGDP is also 5 nM. Equilibrium titrations. A Relative affinity of mant-labeled guanine nucleotide binding to RF3. The ratios of added mant-nucleotide:GDP at half-saturation are indicated dashed lines.
B Competition titrations. Titrations were biphasic, yielding two equilibrium binding constants that were similar for the two complexes. The binding of GTP presumably is stabilized as well, but the effect cannot be determined directly because GTP would be hydrolyzed by ribosome-bound RF3.
Inset: logarithmic plot, half-saturation of high-affinity binding is indicated dashed lines. In control titrations without RF3, no signal change was observed. At low concentration of GTP, i. A Dependence on GTP concentration.
As nucleotide turnover on RF3, i. On the other hand, our data also show that GTP at equilibrium binds to RF3 with an affinity that is comparable with that of GDP 4-fold higher K d , rather than with a fold higher K d , as reported previously based on data obtained by a non-equilibrium filtration assay Following peptide release, the affinity of GDPNP binding to RF3 is increased by three orders of magnitude owing to slower dissociation. The strong stabilization of the GDPNP-bound complex is in line with a closure of the nucleotide binding pocket that was derived from crystal structures of RF3 on the ribosome 11 , However, binding of RF3 to ribosomes that are not in the post-termination state is a rare event, as most of the time during protein synthesis, RF3 has to compete with the large excess of EF-Tu—GTP—aa-tRNA complexes, which is likely to preclude RF3 binding to the ribosome when a sense codon is exposed in the A site.
Thus, GTP hydrolysis by RF3 on ribosomes that are in functional states other than the post-termination state is probably negligible. Present and previous 21 results suggest the following sequence of events during translation termination Figure 6. Schematic representation of translation termination. The nascent peptide is depicted as colored balls. All these proteins have similar characteristic GTP binding motifs; however, they use the energy of GTP hydrolysis in remarkably different ways.
In comparison, ribosome-activated GTP hydrolysis by RF3 is slow, virtually independent on the functional state of the ribosome and does not require the presence of RF2.
Rather, the timing of GTP hydrolysis appears to depend on the internal clock set by the structure of the GTP binding pocket and its interactions with the ribosome. The divergent evolution of translational GTPases and the mechanisms that cause the different rates of GTP hydrolysis are important issues to be addressed in the future.
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Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Frank Peske , Frank Peske. Oxford Academic. However, at variance with these results, Antoun et al. More recently, it was also reported that GTP hydrolysis by IF2 drives the ribosomes to acquire an elongation-competent conformation and commits them to enter the elongation cycle, whereas blocking the GTPase yields 70S complexes unable to enter elongation Our results demonstrate that these IF2 mutants can perform all functions of wild type wt IF2 in vitro and allow near-normal growth of cells lacking wt IF2.
In vivo complementations were performed essentially as described 24 using an E. This strain was transformed with a pGEX plasmid vector carrying B. The presence of the pGEX carrying B. The presence of B. As mentioned previously, selection for intragenic suppressors of dominant lethality due to the loss of the GTPase activity of IF2 resulting from His residue mutations H in E.
To obtain intragenic suppressors affected in other parts of the molecule, we subjected B. The cells capable of surviving the over-expression in trans of the GTPase-deficient IF2 HY mutant were selected and three lethality-suppressor mutations were identified and characterized. All three displayed a reduced affinity for the ribosomal subunits: two of them SP and EK for the 30S subunit and the other GE for both subunits In particular, our attention was attracted by a remarkable feature of the EK mutant in the IF2-G3 domain; in spite of being totally inactive in GTP hydrolysis, it appeared to be able to support initiation dipeptide formation, an IF2 activity considered to be IF2 GTPase-dependent, at least according to the literature Thus, in this study, we introduced in E.
Also the cells expressing IF2 bearing the EK single mutation continued to grow like those expressing wt IF2 Figure 1 A and without any reduction in the viable counts Supplementary Data in Supplementary information.
Over-expression and 30S binding of wt and mutant E. A Growth of E. The color code is the same as in panel C. The in vitro phenotypes of the IF2 E mutant were analyzed in individual reactions of the translation initiation pathway. In addition to its reduced affinity for the 30S subunit, the IF2 EK mutant was completely inactive in ribosome-dependent GTP hydrolysis, even when offered in excess amounts Figure 2 A or under long time of incubation Figure 2 B.
Because a possible reason for this defect could be a failure in substrate binding, the kinetics of mant-GTP and mant-GDP interaction with wt and mutant IF2 were analyzed by fluorescence stopped-flow experiments. As seen from Figure 3 A, the fluorescence intensity of both mant-nucleotides increases rapidly upon their mixing with either type of IF2 and in both cases the binding kinetics reflected a single step interaction mechanism, the resulting tracings of the fluorescence changes being fitted to a single exponential function.
Experimental details can be found in Supplementary information. In the first case, the fluorescence resonance energy transfer FRET signal generated by the proximity between 30S-bound IF3 Atto acceptor and fluorescein-labeled at position 8 fMet-tRNA donor was followed by fluorescence stopped-flow analysis. IF2-dependent formation of 30S IC. Binding was measured by nitrocellulose filtration. C Schematic representation of the steps involved in the formation of the 30S IC.
The first of the two steps was determined by fluorescence stopped-flow analysis, whereas the locked 30S IC were detected by nitrocellulose filtration.
Wild-type and mutant IF2 were also directly compared for their capacity to stimulate initiation dipeptide formation fMet-Phe in this case and to support overall mRNA translation, two activities regarded as being dependent upon the GTPase of IF2 The somewhat reduced activity displayed by the mutant in these two tests cannot be attributed to its lack of GTPase activity because the same reduction in activity compared with wt IF2 was observed also in fMet-tRNA binding Figure 4 A,B , a translation initiation step in which GTP hydrolysis is not involved.
In vitro activities of wt and mutant IF2. Because in the initiation pathway fMet-tRNA binding to the 30S subunit and subunit association precede both dipeptide formation and mRNA translation as a whole, it can be surmised that the diminished activities of the mutant compared with wt IF2 are not due to the GTPase defect but stem from its reduced affinity for the 30S ribosomal subunit and possibly from the slower 30S ICS association.
The standard method to measure in vitro translation by quantifying the incorporation of a radioactive amino acid precursor into an acid-insoluble product Figure 5 A does not distinguish between faithful and non-faithful translation. Because IF2 plays an important role in determining translational fidelity by selectively recognizing fMet-tRNA and kinetically favoring its binding to 30S subunits 30—32 , it seemed important to ascertain if the GTPase-defective mutant of IF2 is still able to ensure a correct translational start.
Thus, an Enzyme-linked immunosorbent assay with monoclonal antibodies directed against the protein encoded by the mRNA template was used to determine not only the level but also the nature of the translation product. The data presented so far demonstrate that the IF2 EK mutant is capable of promoting all activities performed by wt IF2, at least in vitro.
Because B. This choice was made because DNA sequence differences between the two bacterial infB genes and the availability of species-specific anti-IF2 monoclonal antibodies allow the easy detection of the two infB genes and of their products in the cells. Thus, the aforementioned E. Colonies that had lost the vector encoding E. The E. Growth curves in LB of E. The extracts were obtained from saturated cultures, and the individual slots were loaded with increasing amounts of the corresponding extracts from bottom to top, slots 1—6 contain 0.
The slot-blotted filters were exposed to monoclonal antibodies directed against E. However, our results demonstrate that not even a trace amount of wt IF2 is present in the test cells.
In fact, PCR analysis using species-specific primers clearly indicated that these E. Taken together, these results demonstrate that the E. To determine whether in bacteria translation initiation could function efficiently in the absence of the free energy generated by the IF2-dependent hydrolysis of GTP, the properties of two structurally and functionally equivalent IF2 mutants carrying a single amino acid substitution EK and EK in E.
The different phenotypes of the HS and EK mutants, which have in common the inactivation of the GTPase, can be explained by the fact that both are conformational mutants unpublished data from our laboratory but that the conformations resulting from the amino acid substitutions are different and yield proteins with functionally different characteristics.
Furthermore, the IF2 EK mutant was shown to support in vitro all the partial reactions of translation initiation, as well as faithful mRNA translation Figures 4 and 5. Figure 5 C are likely necessary to overcome this defect. The data presented here also demonstrate that a B. However, our results demonstrate that even without the free energy generated by IF2-dependent hydrolysis of GTP, protein synthesis can proceed normally and faithfully, both in vitro and in vivo , despite the fact that the IF2 contribution to translational fidelity 30—32 was reported to be sensitive to the nature of its ligand GTP, GDP or none Indeed, suppressor mutants having these expected properties were found in yeast 36 , 37 and in both B.
Nevertheless, some important structural and functional differences between the yeast and the bacterial system should be mentioned. In contrast, the bacterial phenotype resulting from the expression of GTPase-inactive IF2 mutants is much more severe, causing cell lethality and the intragenic suppressors identified so far have a 7—fold lower affinity either for the ribosomal subunits Figure 1 B or for the acceptor end of fMet-tRNA 23 , 25 and unpublished results.
In conclusion, in light of the present results and of what is known from the literature, it is tempting to draw a parallel between the eukaryotic aIF5B—eIF1A and the bacterial IF2-fMet-tRNA interplay in modulating, together with the guanine nucleotide ligand, the ribosomal affinity of the respective initiation factors.
As to the molecular basis of the phenotypes displayed by the EK and EK mutants, this can be traced back to a structural alteration caused by these substitutions in G3 , which are expected to prevent hydrogen bonding with the backbone amide of G or G in G2 Figure 1 C and D , thereby causing the disruption of the correct G2—G3 interdomain communication and likely constraining the factor into a GDP-like conformation.
Both structural and energetic convergence was found to be quite satisfactory and no systematic hysteresis effects were found. Error bars in Table 1 are given as s.
Axial oxygen ligands to the metaphosphate were restrained to a 2. These reference reactions in solution were run using the same protocol as above.
For reaction 1 weak harmonic restraints to the crystallographic positions with a force constant of 1. Spontaneous activation of the His84 sidechain was monitored by running several independent MD simulations starting from the structure of ribosome bound EF-Tu ternary complex 8 , but with His84 in the inactive conformation that it has in the free ternary complex The p K a shift between active and inactive conformations was calculated from their respective electrostatic free energy differences between the neutral and protonated forms utilizing the linear response approximation 33 ,.
Here, denotes the average electrostatic interaction energy difference between protonated and unprotonated forms of His84 and the subscripts denote that MD sampling is carried out for both protonation states.
For structural comparisons with the experimental data an electron density map was calculated using standard procedures. The coordinate files were hand edited to produce one large coordinate file and a structure factor calculation made using a bulk-solvent correction and isotropic scaling 34 with the Phenix system These calculations produced R work and R free values of 0.
How to cite this article: Wallin, G. Energetics of activation of GTP hydrolysis on the ribosome. Schmeing, T. What recent ribosome structures have revealed about the mechanism of translation.
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