Thermodynamics of GTP and GDP Binding to Bacterial Initiation Factor 2 Suggests Two Types of Structural Transitions

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 °C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 °C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (− 868 ± 25 and − 577 ± 23 cal mol^− 1 K^− 1, respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the γ-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.     

ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis

Summary The inhibition of elongation factors G, Tu and Ts by ppGpp was studied in vitro in a translation system with missense frequency and elongation rate similar to those in vivo. ppGpp inhibits EF-G with K_I=6x10^-5 M. When ppGpp is in twofold excess over GTP and EF-G is the rate-limiting component, the elongation rate is reduced two-fold by ppGpp. EF-Tu is inhibited with K_I=7x10^-7 M in the absence of EF-Ts. When EF-Ts is added, the binding of ppGpp to EF-Tu becomes successively weaker. 1/K_I depends linearly on 1/[Ts] and the intercept at the abscissa gives K_I=4x10^-5 M. This reflects the binding of ppGpp to the binary TuTs complex. The slope reveals that the binding of EF-Ts to the TuMS binary complex is strong (10^-6 M). ppGpp may thus inhibit the cycling of EF-Tu indirectly by the removal of the free EF-Ts by its adsorption to TuMS, as well as directly by simple binding to Tu. EF-Tu inhibition by ppGpp can be fully reversed by high levels of aminoacyl-tRNA only in the presence of EF-Ts and at low ribosomal activity. Our in vitro observations have been extrapolated to in vivo conditions with conclusions as follows: Under strong amino acid starvation ppGpp in two-fold excess over GTP cannot reduce significantly the elongation rate of ribosomes and thereby restore the errors to their normal levels as in the stringent response. Under weak starvation, in contrast, a significant rate reduction can be achieved by the trapping of EF-Ts in complex with TuppGpp.     

Directional transition from initiation to elongation in bacterial translation

The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNA^fMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNA^fMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation.     

Specific interaction of initiation factor IF2 of E. coli with formylmethionyl-tRNA f Met.

The interaction of E.coli initiation factor IF2 with formylmethionyl-tRNA_f^Met has been studied by measuring the inhibition by IF2 of the spontaneous deaminoacylation of the charged tRNA. We find that IF2 protects fMet-tRNA_f^Met against spontaneous deacylation. The formylation is an absolute requirement for this protection and no effect of GTP was found. The association constant for IF2 binding to fMet-tRNA_f^Met at 37°C and physiological ionic conditions was estimated at about 10⁶ M^?1.     

The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits

Ribosomal “stalk” protein L12 is known to activate translational GTPases EF-G and EF-Tu, but not much is known about its role in relation to other two translational G factors, IF2 and RF3. Here, we have clarified the role of L12 in IF2-mediated initiation of bacterial protein synthesis. With fast kinetics measurements, we have compared L12-depleted 50S subunits with the native ones in subunit association, GTP hydrolysis, P_i (inorganic phosphate) release and IF2 release assays. L12 depletion from 50S subunit slows the subunit association step significantly (∼ 40 fold) only when IF2·GTP is present on the 30S preinitiation complex. This demonstrates that rapid subunit association depends on a specific interaction between the L12 stalk on the 50S subunit and IF2·GTP on the 30S subunit. L12 depletion, however, did not affect the individual rates of the subsequent steps including GTP hydrolysis on IF2 and P_i release. Thus, L12 is not a GTPase activating protein (GAP) for IF2 unlike as suggested for EF-G and EF-Tu.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.     

The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNA^fMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNA^fMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNA^fMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNA^fMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNA^fMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNA^fMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.     

Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel_Mtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel_Mtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel_Mtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel_Mtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel_Mtb point mutants that were either synthetase dead (Rel_Mtb^H344Y) or hydrolase dead (Rel_Mtb^H80A). M. tuberculosis strains expressing the synthetase-dead Rel_Mtb^H344Y mutant did not persist in mice, demonstrating that the Rel_Mtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel_Mtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel_Mtb mutant, Rel_Mtb^H80A, that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel_Mtb^H80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel_Mtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.     

Ribosomal Interaction of Bacillus stearothermophilus Translation Initiation Factor IF2: Characterization of the Active Sites

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive “cross-talking” between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.     

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.     

Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg²⁺. We show that (i) eRF3 binds GDP (K_d = 1.9 μM) and this interaction depends only minimally on the Mg²⁺ concentration; (ii) GTP binds to eRF3 (K_d = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg²⁺ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg²⁺, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Chaperone properties of bacterial elongation factor EF-G and initiation factor IF2

Elongation factor G(EF-G) and initiation factor 2 (IF2) are involved in the translocation of ribosomes on mRNA and in the binding of initiator tRNA to the 30 S ribosomal subunit, respectively. Here we report that the Escherichia coli EF-G and IF2 interact with unfolded and denatured proteins, as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-G and IF2 promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with unfolded proteins such as reduced carboxymethyl alpha-lactalbumin. Furthermore, the EF-G and IF2-dependent renaturations of citrate synthase are stimulated by GTP, and the GTPase activity of EF-G and IF2 is stimulated by the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. The concentrations at which these chaperone-like functions occur are lower than the cellular concentrations of EF-G and IF2. These results suggest that EF-G and IF2, in addition to their role in translation, might be implicated in protein folding and protection from stress.     

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt

Mammalian mitochondrial initiation factor 3 (IF3_mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3_mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K_d for the binding of IF3_mt to 28S subunits is ~30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3_mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K_d = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K_d = 0.12−0.25 μM). IF3_mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3_mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3_mt from the 28S subunits upon 39S subunit joining.     

Control of protein synthesis in Escherichia coli: Lack of correlation with changes in intracellular pools of ATP,GTP, and ppGpp

Intracellular pools of ATP, GTP, and ppGpp have been measured in Escherichia coli after an energy source shift-down from glucose minimal to succinate-minimal medium. In a Tic⁺ strain (ATCC 10798), which reduces translational initiation after the down-shift, the rate of protein labeling falls to about 30% of its preshift rate within the first minute after shift and reaches a minimum of 17% by 6 min after shift. The ATP pool in this strain remains constant for about 10 min after shift, then declines gradually to about 60% of its initial level. The temporal discrepancy between protein synthesis and the decline in the ATP pool indicates that a decrease in intracellular ATP is not necessary for the control of protein synthesis. In a Tic^? strain (W1), which cannot control translational initiation under these conditions, the decline in the ATP pool is somewhat more rapid and more pronounced (to 40%) than in the Tic⁺ strain, indicating that the decline in the ATP pool is not sufficient to trigger control of translational initiation. The intracellular GTP pool in the Tic⁺ strain remains constant for 2 min after shift, then declines gradually to reach a minimum of 45% of its initial level at 20 min after shift. The pattern is in general similar in the the Tic^? strain, although the ultimate decline in GTP is more pronounced (to 29%). These data indicate that the decline in GTP is not sufficient and probably unnecessary to elicit control of translational initiation. Intracellular levels of ppGpp increase with very similar kinetics in relA⁺Tic⁺ (ATCC 10798) and relA⁺Tic^? (W1) strains, indicating that elevated ppGpp levels are not sufficient to elicit control of translation. In a relA^?Tic⁺ strain (NF162), or in a relA⁺Tic⁺ strain treated with rifampin, the ppGpp pool does not increase significantly after shift-down although translational initiation is reduced. Thus, an increase in the ppGpp pool is not necessary to control of translational initiation.     

Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC₅₀ of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.     

ppGpp cycle in Escherichia coli.

Summary Kinetics of accumulation and degradation of ppGpp and pppGpp were analysed in spoT ⁺ and spoT strains of Escherichia coli. The experimental data in this paper indicate that on degradation ppGpp is not converted to pppGpp but instead is converted to GDP which is in turn phosphorylated to GTP. In addition the data are consistent with the idea the pppGpp is a direct precursor of ppGpp. We propose that ppGpp is metabolised according to the following pathway: GTP-pppGpp-ppGpp-GDP-GTP, which we call the ppGpp cycle. Coupled with the observations in spoT strains we assume that ppGpp blocks its own synthesis by inhibiting the synthesis of pppGpp but not the interconversion of the two nucleotides.     

Kinetic characterization of recombinant human cytosolic phosphoenolpyruvate carboxykinase with and without a His10-tag

We report the first kinetic characterization of human liver cytosolic GTP-dependent phosphoenolpyruvate carboxykinase (GTP-PEPCK), which plays a major role in the development of type 2 diabetes in human. In this work two recombinant forms of the enzyme were studied. One form had a His₁₀-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn²⁺ was used as the sole divalent cation, the His₁₀-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn²⁺ concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn²⁺ concentration range the His-tagged human PEPCK behaved almost identically to the tag-free enzyme. This property will bring simplicity and speed to purifying and studying multiple structural variants of this important enzyme. Apparent K_m values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 μM, respectively, while those for oxaloacetate and GTP were 4 and 23 μM, respectively, emphasizing the enzyme's gluconeogenic character. Bicarbonate (> 100 mM) inhibited OAA-forming activity, which was a new observation with a GTP-PEPCK. The apparent K_m for Mn²⁺ in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn²⁺ and bicarbonate or CO₂ might regulate the enzyme in vivo.