Mutant Elongation Factor Tu Research Articles

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

Utilizing elongation factor Tu mutagenesis to incorporate large unnatural amino acids in vivo

Site‐specific in vivo incorporation of unnatural amino acids (UAAs) via aminoacyl tRNA synthetase (RS) mutagenesis has been widely used in protein structure and function studies. While a large, diverse family of UAAs has been incorporated into proteins, aminoacyl tRNA synthetases for certain UAAs seem problematic to evolve or generate. We developed a reporter system to explore substrate specificity of current UAA RSs. Using these permissive RSs, structural limitations have been explored. The photocrosslinking UAAs anthraquinonylalanine and 3‐(3’‐fluorenyl‐9’‐oxo)‐alanine should be incorporated by permissive RSs. However, incorporation of these UAAs into proteins has not yet been accomplished. It is the current understanding that elongation factor Tu (EF‐Tu) size limitation is preventing the incorporation of these UAAs. It is believed that UAAs that were previously too large to see incorporation with mutant RSs alone will hopefully be incorporated utilizing mutant RS and mutant EF‐Tu combinations in vivo. Here we report progress with mutant EF‐Tu and orthogonal aminoacyl tRNA synthetase pairs. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364).

Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Mechanism of EF-Ts-Catalyzed Guanine Nucleotide Exchange in EF-Tu: Contribution of Interactions Mediated by Helix B of EF-Tu

Elongation factor Tu (EF-Tu) belongs to the family of GTP-binding proteins and requires elongation factor Ts (EF-Ts) for nucleotide exchange. Crystal structures suggested that one of the salient features in the EF-Tu x EF-Ts complex is a conformation change in the switch II region of EF-Tu that is initiated by intrusion of Phe81 of EF-Ts between His84 and His118 of EF-Tu and may result in a destabilization of Mg2+ coordination and guanine nucleotide release. In the present paper, the contribution of His84 to nucleotide release was studied by pre-steady-state kinetic analysis of nucleotide exchange in mutant EF-Tu in which His84 was replaced by Ala. Both intrinsic and EF-Ts-catalyzed nucleotide release was affected by the mutation, resulting in a 10-fold faster spontaneous GDP release and a 4-fold faster EF-Ts-catalyzed release of GTP and GDP. Removal of Mg2+ from the EF-Tu x EF-Ts complex increased the rate constant of GDP release 2-fold, suggesting a small contribution to nucleotide exchange. Together with published data on the effects of mutations interfering with other putative interactions between EF-Tu and EF-Ts, the results suggest that each of the contacts in the EF-Tu x EF-Ts complex alone contributes moderately to nucleotide destabilization, but together they act synergistically to bring about the overall 60,000-fold acceleration of nucleotide exchange in EF-Tu by EF-Ts.

Qβ-Phage Resistance by Deletion of the Coiled-coil Motif in Elongation Factor Ts

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.

GE2270A-resistant Mutations in Elongation Factor Tu Allow Productive Aminoacyl-tRNA Binding to EF-Tu·GTP·GE2270A Complexes

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E.coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3′ end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu·GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu·GDP·GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu·GTP·GE2270·aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Mutant EF-tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu·GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu·GDP·enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu·GDP·enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu·GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA Trp-derived suppressor, but not wild-type tRNA Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.

Isolation and functional analysis of histidine-tagged elongation factor Tu.

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.

Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough.

This is the first report of ribosomal frameshifting promoted by mutants of the elongation factor Tu (EF-Tu). EF-Tu mutants can suppress both -1 and +1 frameshift mutations. The level of nonsense readthrough is also increased at some UGA (this paper) and UAG (Hughes, 1987) sites by these mutants. Suppression occurs when a mutant tuf allele is paired with a wild-type copy of the other tuf gene but is most efficient when both tuf genes are mutant. Frameshifting mediated by the tuf alleles studied, tufA8 and tufB103, is not general; indeed most frameshift mutations are not suppressed. Several possible mechanisms by which mutant EF-Tu may cause frameshifting are discussed.

A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein.

A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis. The mutated tufA gene was then expressed in maxicells. The properties of [35S]methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays. The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate. The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM). These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring. Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate. Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins. This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins.

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

An Escherichia coli mutant with an altered elongation factor Tu.

A thermosensitive mutant of Escherichia coli has been isolated that is unable to replicate the bacteriophage MS2 at 42 degrees but permits phage production at 37 degrees . Thermal inactivation studies of the supernatant enzymes show that this mutant contains a factor essential for the polymerization of phenylalanine from phenylalanyl-tRNA that at 50 degrees is more rapidly inactivated than the corresponding wild-type factor. The elongation factor Tu (EF-Tu) was isolated and purified to apparent homogeneity as the EF-Tu.GDP complex, both from mutant and wild-type cells. Addition of purified wild-type EF-Tu.GDP to reaction mixtures fully restored the activity of thermally inactivated mutant supernatants. These experiments excluded EF-Ts as the thermolabile factor involved. Similar inactivation studies, dealing with the purified factors and performed in reaction mixtures that were not supplemented with GDP, revealed that the half-life of mutant EF-Tu.GDP at 50 degrees was 1.5 min, that of the wild-type factor 6 min. Addition of GDP (10muM) to the medium reduced the inactivation rate of both wild-type and mutant factor and also the difference in inactivation kinetics. Besides the altered elongation factor Tu, the mutant skill contains a second mutation affecting the glutaminyl-tRNA synthetase.