tufA Gene Research Articles

Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena

The nucleotide sequence of a 6156 bp segment of the circular 73 kb DNA from Astasia longa resembling the chloroplast DNA of Euglena was determined. The genes for the plastid elongation factor Tu (tufA) and the ribosomal protein S7 (rps7), six tRNA genes (trnQ, trnS, trnG, trnM, trnT, trnR), and three open reading frames were identified. These genes show a high degree of sequence similarity (73%-99%) to the corresponding genes on the Euglena chloroplast genome. The tufA gene contains two small AT-rich introns within its coding region. Northern analysis revealed the in vivo transcription of the tufA gene and of a reading frame of 456 codons into monocistronic mRNAs of 1.3 and 1.4 kb, respectively. The arrangement and organization of the genes on the 73 kb DNA of the colourless heterotrophic flagellate Astasia and the chloroplast DNA of autotrophic Euglena are compared.

Expression in Escherichia coli of the tuf Gene from the Extremely Thermophilic Eubacterium Thermotoga maritima: Purification of the Thermotoga Elongation Factor Tu by Thermal Denaturation of the Mesophile Host Cell Proteins

Summary The gene for the elongation factor Tu (EF-Tu) of the extemely thermophilic eubacterium Thermotoga maritima was identified by cross-hybridization with the tufA gene of Escherichia coli and cloned into plasmid pBR322. The tuf gene of T. maritima was found to be present in a single copy in the Thermotoga chromosome and to lie only 11 nucleotides downstream of the terminating codon of the EF-G gene. The Thermotoga tuf gene was expressed in E. coli either following transcription from its own promoter or from the trp promoter of plasmid pDR720; the tuf gene expression was demonstrated by assaying the GDP-binding activity surviving heat-treatment (up to 90°C) of host-cell extracts. The formation of the thermostable EF-Tu in E. coli cells was also verified by the presence, in the cell extracts, of a protein specifically recognized by T. maritima anti-EF-Tu antibodies, and having the same relative molecular mass (Mr 49,000), and the same V8 protease cleavage pattern, as authentic Thermotoga EF-Tu. That protein was the predominant species found in the extracts following heating at 65°C and removal of precipitated proteins. The Thermotoga factor expressed in E. coli was unable to bind GDP at 37°C, a heat-treatment at high temperatures being required to overcome conformational barriers that hinder the proper folding of the polypeptide.

Determination of the number oftufgenes inChlamydia trachomatisandNeisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.

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)

Duplication of the tuf gene: a new insight into the phylogeny of eubacteria

The conservation and duplication of the tuf gene encoding the elongation factor EF-Tu were used to define phylogenetic relationships among eubacteria. When the tufA gene of Escherichia coli was used as a probe in hybridization experiments, duplicate tuf genes were found in gram-negative bacteria from three major phyla: purple bacteria, bacteroides, and cyanobacteria. Only a single copy of tuf was found in gram-positive bacteria, including mycobacteria and mycoplasmas. Gram-positive clostridia were found to carry two copies of tuf.

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.

Effects of GUG and AUG initiation codons on the expression of lacZ in Escherichia coli

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.

Precursor for elongation factor Tu from Escherichia coli.

The tufA gene, one of two genes in Escherichia coli encoding elongation factor Tu (EF-Tu), was cloned into a ColE1-derived plasmid downstream of the lac promoter-operator. In cells carrying this plasmid, the synthesis of EF-Tu was increased four- to fivefold upon the addition of isopropyl-beta-D-thiogalactopyranoside (an inducer of the lac promoter). This condition led to the synthesis of a novel protein, called pTu, which comigrated with EF-Tu on a sodium dodecyl sulfate-polyacrylamide gel but could be separated on an isoelectric focusing gel, since pTu is slightly more basic than EF-Tu. The synthesis of pTu could also be induced by the synthesis of a hybrid protein containing just the amino-terminal half of the EF-Tu protein. Genetic data suggest that pTu is the product of the tufA and tufB genes. The pTu protein was shown to be related to EF-Tu by gel electrophoresis of tryptic peptides. Pulse-chase experiments suggest that pTu is a precursor of EF-Tu. Interestingly, in a classic membrane fractionation procedure, EF-Tu was found in the cytosolic fraction, whereas pTu was partitioned with the outer membrane.

The nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G.

We have determined the nucleotide sequence of the Escherichia coli fus gene, which codes for elongation factor G. The protein product of the sequenced gene contains 703 amino acids, with a predicted molecular weight of 77,444. The fus gene shows the nonrandom pattern of codon usage typical of ribosomal proteins and other proteins synthesized at a high level. We have identified several potential promoter sequences within the gene. One of these sequences may correspond to the secondary promoter for expression of the downstream tufA gene (encoding elongation factor Tu) whose activity has been described previously (1,2). A comparison of the nucleotide and amino acid sequences of elongation factors G and Tu reveals a limited but significant homology between the two proteins within the 150 amino acid residues at their amino-terminal ends.

Tuf gene dosage effects on the intracellular concentration of EF-TuB.

In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL) of phage lambda. Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL. Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 28 degrees C to 37 degrees C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF-Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37 degrees C to 43 degrees C. The multicopy plasmid pTuB1 described by Miyajima et al. [FEBS Lett. 102, 207-210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB. Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.

Location of the single gene for elongation factor Tu on the Euglena gracilis chloroplast chromosome.

The structural gene for elongation factor Tu (EF-Tu) has been mapped by heterologous hybridization to a 2900 base pair sequence of Euglena gracilis Klebs Strain Z Pringsheim chloroplast DNA within the EcoRI fragment, Eco N. The hybridization probes were obtained from a HhaI restriction fragment containing internal sequences to Escherichia coli EF-Tu, located in the tufA gene locus, and from an EcoRI restriction fragment of chloroplast DNA from the eukaryotic algae Chlamydomonas reinhardii, containing the 3' end of the chloroplast EF-Tu gene. This is the second identified protein gene locus to be mapped on the E. gracilis chloroplast genome, and the first using prokaryotic DNA as a probe.

Rates of growth, ribosome synthesis and elongation factor synthesis in a tufA defective strain of E. coli.

A tufA defective strain of E. coli was isolated which by a single deletion event acquired a tufA-lacZ fusion gene and lost the normal functional tufA gene (see accompanying paper). A correlation between the growth rate of protein synthesis was decreased to about 50% in the tufA defective strain whereas the number of EF-Tu molecules per ribosome was about 80% compared to a normal strain. The results indicate that tufB gene expression was preferentially stimulated in the tufA defective strain but the increased EF-TuB synthesis was not sufficient to make up for the loss of normal EF-TuA synthesis. Introduction of a plasmid that carries a complete tufA gene and the preceeding fusA gene but not the str-promotor into the tufA defective strain did not alleviate the slow growth or low rate of EF-Tu synthesis showing that the high rate of EF-TuA synthesis compared to the other proteins in the str operon is not augmented by a strong second promotor for the tufA gene. The tufA-lacZ fusion which takes the place of the normal tufA gene was expressed at a high rate and the beta-galactosidase activity increased with the growth rate as expected.

Construction and characterization of a tufA-lacZ fusion coding for an E. coli EF-Tu-beta-galactosidase chimeric protein.

A new phage lambda cloning vector was constructed that has a single EcoRI site upstream from weakly expressed lacI-Z gene isolated by Müller-Hill and Kania (1974). An EcoRI fragment containing the complete tufA gene of E. coli was cloned on the vector and the recombinant phage was crossed into the str operon that has tufA as its last gene. Subsequent selection gave rise to a tufA-lacZ fusion that codes for a chimeric peptide. The fused peptide has a molecular weight of 148,000 and contains 40% of the N-terminal of EF-Tu followed by part of the lac repressor-beta-galactosidase fusion. The specific activity of the fused peptide is about half of the activity of normal beta-galactosidase.

Regulation of the synthesis of E. coli elongation factor TU

Rapidly growing E. coli with two active genes (tufA, tufB) for elongation factor (EF) Tu contains three times as much EF-Tu (tuf) mRNA as EF-G (fus) mRNA on a molar basis, but about seven times as much EF-Tu as EF-G or ribosomes. The concentration and translational efficiency of fus (EF-G) mRNA is about that for ribosomal protein mRNAs. The high molar concentration of EF-Tu relative to EF-G or ribosomes is achieved in part by translating tuf mRNA more efficiently than these other mRNAs. In a tufA + tufB -::Mu strain, the tuf:fus mRNA ratio is 1, but the concentration of EF-Tu and tuf mRNA is the same as in the wild-type strain. Thus cells with only an active tufA gene increase the concentration but not the translational efficiency of tuf mRNA. In such cells the concentration of fus mRNA is almost three times that in the wild-type strain. Because the tufA gene is distal to but cotranscribed with the fus gene as part of the four gene str operon, the wildtype concentration of tuf mRNA in these tufB - cells must be produced by increasing the concentration of transcript corresponding to the entire str operon. Thus transcription of the tufA gene can only proceed from the str promoter. Extracts of the tufBcells contain tuf transcripts that correspond not only in size to the entire 4.5 kb str operon, but also to the size (≈1 kb) of a tuf gene. Our evidence suggests that this 1 kb tuf transcript is derived by posttranscriptional modification of the primary str operon transcript and that this modification could in part explain the high translational efficiency of tuf mRNA.

Molecular properties of two mutant species of the elongation factor Tu.

The molecular properties of two mutant species of the elongation factor Tu (EF-Tu), derived from either tuf A or tuf B, have been studied. One, designated EF-TuAR, is the product of a kirromycin-resistant tufA gene. The other designated EF-TuBO is a tuf B product and is present in a kirromycin-resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF-TuAR species. EF-TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tuf B gene has been inactivated by an insertion of the bacteriophage Mu. EF-TuBO has been isolated from LBE 2012 together with EF-TuAR in a 1:1 mixture. Fractionation of this mixture of DEAE-Sephadex A-50 resulted in an enrichment of EF-TuBO of about 80%. The properties of EF-TuAR and EF-TuBO have been compared to those of a kirromycin-sensitive species designated EF-TuAS, which was isolated from LBE 2045 by transduction of wild-type tuf A. We show here that all three EF-Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF-Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF-TuAS (wild-type tuf A gene product) to be retained on, and thus block, the ribosome. (b) EF-TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EF-TuBO fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF-TuAR (LBE 2012) is kirromycin resistant.

The nucleotide sequence of the cloned tufA gene of Escherichia coli

The 4 kb (8.5 %λ units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G ( fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

Q beta replicase containing wild type and mutant tufA and tufB gene.

The protein synthesis elongation factor EF-Tu, complexed with EF-Ts, forms part of Q beta RNA replicase. In an effort to determine its function in the RNA synthesis reaction, we have developed procedures which allow us to replace the endogenous EF-Tu in purified Q beta replicase with EF-Tu from a variety of sources. In this communication we report purification of EF-Tu from strains containing (a) a wild type tufA gene only, (b) a kirromycin-resistant mutant tufA gene only, and (c) a kirromycin-resistant mutant tufA gene and a mutant tufB gene which codes for EF-Tu that does not bind ribosomes. When each of these EF-Tu preparations is inserted in Q beta replicase, the wild type tufA gene product and and the tufB gene product function appearently normally, but the kirromycin-resistant tufA gene product causes the formation of an altered enzyme. The Q beta replicase containing kirromycin-resistant EF-Tu is unstable; it is rapidly inactivated in the reaction mixture, even at temperatures as low as 20 degrees C. This property results in an apparent increase in template specificity; while wild type Q beta replicase will transcribe poly(C) and other synthetic RNA species, the mutant enzyme will do so only in the presence of Mn2+, which reduces template specificity. The kirromycin-resistant Q beta replicase will also transcribe Q beta RNA. The results imply that EF-Tu is involved in maintenance of enzyme structure, which, in turn, is implicated in template specificity.

Studies on Stringent Control in a Cell-Free System

The biosynthesis of elongation factor Tu (EF-Tu) has been studied in a cell-free system with DNA of the transducing phage lambdarifd18 as a template. It was found that the synthesis of EF-Tu in this system was inhibited by about 60% in the presence of 0.3 to 0.6 mM guanosine-5'-diphosphate-3'-diphosphate (ppGpp). The syntheses of several ribosomal proteins encoded in this template, i.e. L1, L10, L11, and L7/L12, were also depressed, whereas those of phage lambda proteins were rather enhanced by the addition of ppGpp. By separating the reaction into two steps, i.e., transcription and translation, the effect of ppGpp was shown to occur at the level of transcription. Several analogs, such as guanosine-5'-diphosphate-3'-monophosphate (ppGp) and guanosine-5'-diphosphate (ppG), were without effect. The formation of mRNA for EF-Tu was assessed directly by specific hybridization with pTUA1 DNA carrying tufA gene. The results clearly indicated that the synthesis of tufB . MRNA was severely and selectively inhibited by ppGpp.

Cloning of an EcoRI fragment carrying E. coli tufA gene

EcoRI fragments of the transducing phage lambda fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Apr) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and is EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA . DNA hybridization of RNA transcribed from phage lambda rifd18 carrying tufB with DNA from pTUAl.