Mutant EF-Tu Research Articles

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

Temperature-Sensitive Mutants of RNase E in Salmonella enterica

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

Mutants of the RNA‐processing enzyme RNase E reverse the extreme slow‐growth phenotype caused by a mutant translation factor EF‐Tu

Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n= 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n= 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

Functional EF-Tu with large C-terminal extensions in an E. coli strain with a precise deletion of both chromosomal tuf genes

An Escherichia coli strain was constructed in which both chromosomal genes encoding elongation factor (EF)-Tu (tufA and tufB) have been inactivated with precise coding sequence replacements. A tufA gene in an expression vector is supplied as the sole EF-Tu source. By using plasmid replacement, based on plasmid incompatibility, mutant EF-Tu variants with a large C′-terminal extension up to 270 amino acids were studied and proved to be functional in a strain lacking the chromosomal tufA and tufB genes.

Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu.

Several elongation factor (EF) Tu mutants (T25A, H22Y/T25S, D80N, D138N) that have impaired nucleotide binding show decreased solubility on overexpression in the E. coli cell, an indication that they do not fold correctly. Moreover, EF-Tu[T25A] and EF-Tu[D80N] were shown to inhibit cell growth on expression, an effect attributed to their sequestration of EF-Ts [Krab, I. M., and Parmeggiani, A. (1999) J. Biol. Chem. 274, 11132--11138; Krab, I. M., and Parmeggiani, A. (1999) Biochemistry 38, 13035--13041]. We present here results showing that the co-overexpression of EF-Ts at a 1:1 ratio dramatically improves the solubility of mutant EF-Tu, although in the case of EF-Tu[D138N]--which cannot at all bind the nucleotides available in the cell--this is a slow process. Moreover, with co-overexpression of EF-Ts, the mentioned growth inhibition is relieved. We conclude that for the formation of a correct EF-Tu structure the nucleotide plays an important role as a "folding nucleus", and also that in its absence EF-Ts can act as a folding template or steric chaperone for the correct folding of EF-Tu.

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.

Translational Activities of EF-Tu [G222D] Which Cannot Be Reconciled with the Classical Scheme of the Polypeptide Chain Elongation Cycle

We have developed a cell-free system ofE. colithat enables us to study thein vitrotranslation of natural mRNA mediated by wild-type or mutant EF-Tu. Various mutant EF-Tu species have been analyzed, one of which, EF-Tu [G222D], appeared to be virtually unable to mediate the translation of natural mRNA. Since this mutant factor is able to participate in translationin vivoby suppressing nonsense and frameshift mutations in cooperation with EF-Tu [A375T], a revision of the generally accepted scheme of the elongation cycle has been proposed (Bosch, L., Vijgenboom, E., & Zeef, L.A.H., 1996,Biochemistry36).

Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli.

Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT mutations cluster in the three-domain junction interface of EF-Tu. GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.

Histidine-118 of elongation factor Tu: its role in aminoacyl-tRNA binding and regulation of the GTPase activity

The function of His 118 in elongation factor (EF)-Tu from Escherichia coli was investigated by its substitution with glycine. The substitution had a differential effect on individual functions of the protein. The affinity for aminoacyl (aa)-tRNA and the intrinsic GTPase activity of the mutant EF-Tu were decreased whereas the response of its GTPase center to aa-tRNA was strongly increased. These results suggest that the region around His 118 is involved in the binding of aa-tRNA and in the transmission of a turn-off signal generated by the interaction with aa-tRNA and directed to the GTPase center of EF-Tu.

Mutant ribosomes can generate dominant kirromycin resistance

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.

Translational Frameshifts Induced by Mutant Species of the Polypeptide Chain Elongation Factor Tu of Escherichia coli

Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli. Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu. A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting. The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately. These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.

Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors.

Read-through of nonsense codons has been studied in wild-type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF-Tu. The two phenomena differ essentially. Readthrough of UGA in wild-type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid-borne EF-Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF-Tu. Strains of E. coli harbouring mutant species of the elongation factor EF-Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF-Tu species: EF-TuAR(Ala 375----Thr) and EF-TuBo(Gly 222----Asp). Cells harbouring EF-TuAR(Ala 375----Thr) and wild-type EF-TuB, or wild-type EF-TuA and EF-TuBo(Gly 222----Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF-Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF-Tu molecules on the ribosome.

Altered regulation of the guanosine 5'-triphosphate activity in a kirromycin-resistant elongation factor Tu.

In the preceding article a mutant elongation factor Tu (EF-TuD2216) resistant to the action of kirromycin was found to display a spontaneous guanosine 5'-triphosphatase (GTPase) activity, i.e., in the absence of aminoacyl transfer ribonucleic acid (tRNA) and ribosome-messenger RNA. This is the first example of an Ef-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. In this study we show that this activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation caused an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity an increased affinity for GTP are two properties also found with wild-type EF-Tu in the presence of kirromycin [Fasano, O., Burns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565; Sander, G., Okonek, M., Crechet, J.-B., Ivell, R., Bocchini, V., & Parmeggiani, A. (1979) FEBS Lett. 98, 111-114]. Therefore, both binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216.GDP but still does with EF-TuD2216.GTP in agreement with our previous finding that EF-Tu.GTP is the preferential target of the antibiotic in the wild type [Fasano, O., Bruns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565). The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome.mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(phenyl-alanine/leucine) synthesis at increasing Mg2+ concentrations, is identical for both wild-type and mutant EF-Tu.

Interaction of host-coded and virus-coded polypeptides in RNA phage replication.

The enzymes responsible for replication of the RNA of the single-stranded RNA bacteriophages contain, in addition to one phage-coded polypeptide, three host-coded polypeptides taken from the protein biosynthetic machinery: ribosomal protein S1 and the elongation factors Tu and Ts. While S1 performs a function in RNA replication derived from its protein synthetic function, mRNA binding, the reactions catalysed by the elongation factors in protein synthesis are apparently dispensible for RNA replication. In the replicase, these polypeptides, acting as the EF-Tu . Ts complex, play a fundamental structural role. Replacement of the endogenous EF-Tu with mutant EF-Tu, itself stable, causes the RNA replicase to become unstable. The possibility that EF-Tu . Ts is solely a structural protein in the RNA replicase is suggested by experiments showing that a variety of modifications of the elongation factors can be tolerated without loss of RNA synthetic capacity. In fact, EF-Tu . Ts from distantly related bacterial species can substitute for E. coli EF-Tu . Ts in RNA replicase. Evidence is presented that the high in vitro template specificity of Q beta replicase may be accomplished through modulation of the level of GTP required for initiation of transcription. Different natural and synthetic RNAs require quite different GTP concentrations. Mn2+ ions, which extend the range of templates transcribed by Q beta replicase, lower the requirement for GTP. High ionic strength, which alters the conformation of Q beta replicase such that template specificity is increased, raises the GTP requirement. An additional host coded protein required for in vitro Q beta RNA replication, host factor (HF), interacts specifically with Q beta RNA. This polypeptide acts by allowing Q beta replicase to initiate RNA synthesis with Q beta RNA at reduced GTP concentration.