Both genes for EF-Tu in Salmonella typhimurium are individually dispensable for growth

Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.     

A chimeric disposition of the elongation factor genes in Rickettsia prowazekii.

An exceptional disposition of the elongation factor genes is observed in Rickettsia prowazekii, in which there is only one tuf gene, which is distant from the lone fus gene. In contrast, the closely related bacterium Agrobacterium tumefaciens has the normal bacterial arrangement of two tuf genes, of which one is tightly linked to the fus gene. Analysis of the flanking sequences of the single tuf gene in R. prowazekii shows that it is preceded by two of the four tRNA genes located in the 5' region of the Escherichia coli tufB gene and that it is followed by rpsJ as well as associated ribosomal protein genes, which in E. coli are located downstream of the tufA gene. The fus gene is located within the str operon and is followed by one tRNA gene as well as by the genes secE and nusG, which are located in the 3' region of tufB in E. coli. This atypical disposition of genes suggests that intrachromosomal recombination between duplicated tuf genes has contributed to the evolution of the unique genomic architecture of R. prowazekii.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.     

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.     

The role of EF-Tu and other translation components in determining translocation step size

The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.     

Molecular cloning and sequence determination of the tuf gene coding for the elongation factor Tu of Thermus thermophilus HB8

The tuf gene, which encodes the elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, and its flanking regions were cloned and sequenced. The gene encoding EF-G was found upstream of the 5' end of the tuf gene. The tuf gene of T. thermophilus HB8 had a very high G + C content and 84.5% of the third base in codon usage was either G or C. The deduced primary structure of the EF-Tu was composed of 405 amino acid residues with a Mr = 44658. A comparison of the amino acid sequence of EF-Tu from T. thermophilus HB8 with those of Escherichia coli and Saccharomyces cerevisiae mitochondria showed a very high sequence homology (65-70%). Two Cys residues out of the three found in E. coli EF-Tu had been replaced with Val in T. thermophilus HB8 EF-Tu. An extra amino acid sequence of ten residues, consisting predominantly of basic amino acids (Met-182-Gly-191), which does not occur in EF-Tu of E. coli, was found in T. thermophilus HB8.     

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.     

Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8

The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.     

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.     

The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms.

Nucleotide (nt) sequences encoding the elongation factor Tu (EF-Tu), tRNA(Thr) and tRNA(Trp) from Chlamydia trachomatis have been determined. The environment of the EF-Tu-encoding gene (tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus. The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies. Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies. Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root. The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences. Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein.     

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.     

Divergence among genes encoding the elongation factor Tu of Yersinia Species

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.     

Control of the tRNA-tufB operon in Escherichia coli. 3. Feedback inhibition of tufB expression by an EF-Tu with a deletion in the guanine-nucleotide-binding domain

The expression of tufB, one of the two EF-Tu-encoding genes in Escherichia coli, is under autogenous control. Feedback inhibition of tufB expression by plasmid-borne EF-Tu has been used to answer the question of whether or not the integrity of the guanine-nucleotide-binding domain of EF-Tu is required for the autoregulatory role of the factor protein. We show that a large deletion of tufB, causing the elimination of an 81-amino-acid segment from the plasmid-borne EF-Tu, does not abolish tufB repression. We conclude that the autoregulation of the cellular EF-Tu level is not dependent on an intact guanine-nucleotide-binding domain and does not require binding of GTP to EF-Tu. The repressor activity of the deletion derivative of EF-Tu can be measured despite a rapid disappearance of the (altered) mutant protein from the soluble cytoplasmic fraction of the cell. Degradation and assembly in larger complexes are responsible for this disappearance.     

耐热蛋白质延长因子（EF—Tu）的分子和功能的性质

Protein synthesis elongation factor Tu (EF-Tu) was purified from an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum. The relative molecular mass of EF-Tu. GDP was 51,000. The factor was heat stable and lost only 50% of its activity after heating at 80 degrees C for 5 min. Native and reduced EF-Tu or EF-Tu. GDP contained one SH-reactive group. The elongation factors from C. hydrogenophilum and E. coli were shown to be immunologically identical. From the Southern hybridization analysis seems to suggest that chromosome DNA of C. hydrogenophilum has two tuf genes.     

Predicting the binding affinities of misacylated tRNAs for Thermus thermophilus EF-Tu.GTP

The free energies for the binding of 20 different unmodified Escherichia coli elongator aminoacyl-tRNAs to Thermus thermophilus elongation factor Tu (EF-Tu) were determined. When combined with the binding free energies for the same tRNA bodies misacylated with either valine or phenylalanine determined previously [Asahara, H., and Uhlenbeck, O. C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3499-3504], these data permit the calculation of the contribution of each esterified amino acid to the total free energy of binding of the complex. The two data sets can also be used to calculate the free energy of binding of EF-Tu to any misacylated E. coli tRNA, and the values agree well with previously published experimental values. In addition, a survey of active misacylated suppressor tRNAs suggests that a minimal threshold of binding free energy for EF-Tu is required for suppression to occur.