Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes.

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tumt, show distinct differences in their affinities for guanine nucleotides and in their interactions with elongation factor Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu binds GDP much more tightly than EF-Tumt. This difference does not reside in domain I alone but is regulated by interactions with domains II and III. All the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRNA although some had an increased or decreased activity in this assay. The activity of E. coli EF-Tu but not of EF-Tumt is stimulated by E. coli EF-Ts. The presence of any one of the domains of EF-Tumt in the prokaryotic factor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tumt allowed the mitochondrial factor to interact with bacterial EF-Ts. This observation indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Tsmt interacts with all of the chimeras produced. However, with the exception of domain III exchanges, it inhibits the activities of the chimeras indicating that it could not be productively released to allow formation of the ternary complex. The unique ability of EF-Tumt to promote binding of mitochondrial Phe-tRNAPhe to the A-site of the ribosome resides in domains I and II. These studies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains.     

Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.     

Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.     

Analysis of rate constants governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The kinetics of the heterologous exchange of GDP bound to EF-Tu by free GTP catalysed by EF-Ts have been analysed with a view to correlating results obtainable with different computational procedures. The affinity of EF-Ts for EF-Tu.GTP was found to be somewhat less than previously proposed by Romero et al. (Biochemistry 260, 6167:1985) though still greater than for EF-Tu.GDP. There is a close interrelationship between the constants for the binding of GTP to EF-Tu.EF-Ts and of EF-Ts to EF-Tu.GTP. The declining fractional rate of exchange observed by Romero et al. during displacement of GDP by GTP appears to be dependent on the ratio of the rate constants (k-1 + k-2)k4/k1k-2 as defined in the text, not on that of K4/K1 as they proposed.     

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Isolation,crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.     

Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.     

Determination of the kinetics of guanine nucleotide exchange on EF-Tu and EF-Ts: continuing uncertainties

An analysis is made of the rate constants for the reactions involving the interactions of EF-Tu, EF-Ts, GDP, and GTP recently derived by Gromadski et al. [Biochemistry 41 (2002) 162]. Though their measured values appear to allow a reasonable rate of nucleotide exchange sufficient to support rates of protein synthesis in vivo, their data underestimate the thermodynamic barrier involved in nucleotide exchange and therefore cannot be considered definitive. A kinetic scheme consistent with the thermodynamic barrier can be achieved by modification of various rate constants, particularly of those involving the release of EF-Ts from EF-Tu.GTP.EF-Ts, but such constants are markedly different from what are experimentally observed. It thus remains impossible at present satisfactorily to model guanine nucleotide exchange on EF-Tu, catalysed by EF-Ts by a double displacement mechanism, with experimentally derived rate constants. Metabolic control analysis has been applied to determine the degree of flux control of the different steps in the pathway.     

Interaction of brain transferase I with guanosine nucleotides and aminoacyl-tRNA

Brain transferase I has been purified, and its interactions with GTP and aminoacyl-tRNA have been studied. The data suggest that a transferase I-GTP complex can be formed which interacts with aminoacyl-tRNA to yield an aminoacyl-tRNA-transferase I-GTP complex.     

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome.     

Interaction of bovine mitochondrial ribosomes with messenger RNA

The gene for subunit II of cytochrome oxidase (CoII) from bovine mitochondria has been cloned behind a T7 promoter and the corresponding mRNA synthesized in vitro. The RNA transcribed from this vector has a single nucleotide 5' to the start AUG and, thus, corresponds closely to the native mRNA. It binds to the small 28 S ribosomal subunit of bovine mitochondria but not to the large (39 S) subunit or to 55 S ribosomes. The binding occurs readily in the absence of auxiliary initiation factors or initiator tRNA. The complex formed appears to contain 1 mRNA/28 S subunit. The observed binding is specific for mRNA since neither tRNA nor ribosomal RNA can act as competitive inhibitors. The interaction of the mRNA with the 28 S subunit does not require an AUG codon near the 5' end and constructs containing 5' leaders of more than 100 nucleotides still bind efficiently. About 5% of the bound mRNA is protected from digestion by T1 RNase. The protected fragments do not arise from a specific region of the mRNA since they hybridize to several restriction fragments of the cloned CoII gene.     

Interaction of ras oncogene product p21 with guanine nucleotides

The nucleotide exchange reaction was observed with purified ras oncogene product p21 overproduced in Escherichia coli (Hattori, S. et al. (1985) Mol. Cell Biol. 5, 1449-1455) under various conditions. (NH4)2SO4 increased the rate of dissociation of bound GDP from c-rasH and v-rasH p21. The dissociation kinetics were those of a first order reaction, and there was a linear relationship between the rate constant and the (NH4)2SO4 concentration. At any concentration of (NH4)2SO4, the exchange rate was faster with v-rasH p21 than that with c-rasH p21. EDTA and (NH4)2SO4 synergetically stimulated the dissociation reaction. Nucleotide-free p21 was prepared by gel filtration on Sephadex G-25 in the presence of 5 mM EDTA and 200 mM (NH4)2SO4 at room temperature. The free p21 was quite thermolabile, but the addition of GDP or GTP completely protected p21 from thermal inactivation. The dissociation constants for GDP and GTP were determined with free p21 to be 8.9 and 8.2 nM, respectively, for v-rasH p21, and 1.0 and 2.6 nM for c-rasH p21. In the presence of 200 mM (NH4)2SO4, these dissociation constants increased 3- to 12-fold.     

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.     

Interaction of elongation factor 2 from wheat germ with guanosine nucleotides and ribosomes.

1. The amino acid composition of wheat germ EF2 differs to some extent from that of elongation factors from mammals and bacteria. 2. The purified wheat germ EF2, similarly as the factors from other sources, is active in the: EF1-dependent polymerization of phenylalanine; ribosome-dependent GTP hydrolysis; binding of guanosine nucleotides; and ADP-ribosylation in the presence of diphtheria toxin. Fusidic acid at a concentration of 1 mM inhibits all these EF2-dependent reactions. 3. Diphtheria toxin in the presence of NAD+ inhibits polymerization of phenylalanine but does not effect GTP binding to EF2. 4. Binding of GDP to wheat germ EF2 is inhibited by ribosomes. During interaction with ribosomes, GTP in EF2-GTP complex is rapidly hydrolysed to GDP. Both GTP and 5'-guanylmethylenediphosphonate competitively inhibit formation of the ribosome-EF2-GDP complex due to the replacement of GDP from the complex. The latter is stabilized by fusidic acid.     

Interaction of mammalian mitochondrial ribosomes with the inner membrane

All of the products of mitochondrial protein biosynthesis in animals are hydrophobic proteins that are localized in the inner membrane. Hence, it is possible that the synthesis of these proteins could occur on ribosomes associated with the inner membrane. To examine this possibility, inner membrane and matrix fractions of bovine mitochondria were examined for the presence of ribosomes using probes for the rRNAs. Between 40 and 50% of the ribosomes were found to fractionate with the inner membrane. About half of the ribosomes associated with the inner membrane could be released by high salt treatment, indicating that they interact with the membrane largely through electrostatic forces. No release of the ribosome was observed upon treatment with puromycin, suggesting that the association observed is not due to insertion of a nascent polypeptide chain into the membrane. A fraction of the ribosomes remained with residual portions of the membranes that cannot be solubilized in the presence of Triton X-100. These ribosomes may be associated with large oligomeric complexes in the membrane.     

Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s(-1)) and is accelerated by eEF1Balpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Balpha binding to eEF1A (10(7)-10(8) M (-1) s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.