Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.     

Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain

Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NH2-terminal domain (ezrin 1-233) and that of a COOH-terminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Sf9 cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 microns in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstrom, and A. Vaheri. 1994. J. Cell Biol. 126:1445- 1453), suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NH2- terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.     

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.     

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

Crystals of partially trypsin-digested elongation factor Tu

Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).     

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi. GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9138]. In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu.GDPoxi induces a conformation resembling that of the EF-Tu.GDPoxi complex in the absence of EF-Ts. Binding of EF-Ts to EF-Tu.GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu.GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA.EF-Tu.GTP complex formation.     

DNA-induced alpha-helical structure in the NH2-terminal domain of histone H1

It is important to establish the structural properties of linker histones to understand the role they play in chromatin higher order structure and gene regulation. Here, we use CD, NMR, and IR spectroscopy to study the conformation of the amino-terminal domain of histone H1 degrees, free in solution and bound to the DNA. The NH(2)-terminal domain has little structure in aqueous solution, but it acquires a substantial amount of alpha-helical structure in the presence of trifluoroethanol (TFE). As in other H1 subtypes, the basic residues of the NH(2)-terminal domain of histone H1 degrees are clustered in its COOH-terminal half. According to the NMR results, the helical region comprises the basic cluster (Lys(11)-Lys(20)) and extends until Asp(23). The fractional helicity of this region in 90% TFE is about 50%. His(24) together with Pro(25) constitute the joint between the NH(2)-terminal helix and helix I of the globular domain. Infrared spectroscopy shows that interaction with the DNA induces an amount of alpha-helical structure equivalent to that observed in TFE. As coulombic interactions are involved in complex formation, it is highly likely in the complexes with DNA that the minimal region with alpha-helical structure is that containing the basic cluster. In chromatin, the high positive charge density of the inducible NH(2)-terminal helical element may contribute to the binding stability of the globular domain.     

Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu

Turnip yellow mosaic virus (TYMV) Val-RNA forms a complex with the peptide elongation factor Tu (EF-Tu) in the presence of GTP: the Val-RNA is protected by EF-Tu·GTP from non-enzymatic deacylation and nuclease digestion. The determination of the length of the shortest TYMV Val-RNA fragment that binds EF-Tu·GTP leads us to conclude that the valylated aminoacyl RNA domain equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T arm is sufficient for complex formation. Since the aminoacyl RNA domain is also sufficient for adenylation by the ATP(CTP):tRNA nucleotidyltransferase, an analogy can be drawn between these two tRNA-specific proteins.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

T-armless tRNAs and elongated elongation factor Tu

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.     

Antisuppression by mutations in elongation factor Tu

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.     

Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.

The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively. The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1). At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell.     

tRNA and the guanosinetriphosphatase activity of elongation factor Tu

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Translational regulation by modifications of the elongation factor Tu

EF-Tu fromE. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorlation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys⁵⁶ which is converted to Me₂Lys upon entering the stationary phase. Lys⁵⁶ is in the GTPase switch-1 regions (residues 49–62), a strongly conserved site involved in interactions with the nucleotide and the 5′ end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy.In vivo 5–10% of EF-Tu is phosphorylated at Thr³⁸² by a ribosome-associated kinase. In EF-Tu-GTP, Thr³⁸² in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu¹¹⁷ that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyl tRNA. Thein vivo role of this reversibles modification is still a matter of speculation. T4 infection ofE. coli may trigger a phage-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly⁵⁹-Ile⁶⁰ in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanisms ofin cis inhibition of late T4 mRNA translation. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.