Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.     

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.     

Nucleotide sequence of the gene coding for the elongation factor Tu from the extremely thermophilic eubacterium Thermotoga maritima

Abstract The gene coding for the elongation factor Tu (EF-Tu) of Thermatoga maritima was cloned and sequenced. The predicted amino acid sequence was compared with those of other eubacteria, an archaebacterium and two eukaryotes as well. The similarity values and the distance matrix tree show that Thermotoga is more closely related to the eubacteria than to the representatives of the other urkingdoms. Thermotoga maritima represents the deepest branching within the tree of EF-Tu sequences from all eubacteria studied so far.     

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.     

Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the 'S10 and spectinomycin' operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.     

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.     

Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus

The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

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.     

Overexpression of the methanococcal ribosomal protein L12 in Escherichia coli and its incorporation into halobacterial 50 S subunits yielding active ribosomes

The gene for the ribosomal L12 protein from the archaebacterium Methanococcus vannielii was cloned into the expression vector pKK223-3. The protein was overexpressed and remained stable in Escherichia coli XL1 cells. Purification yielded a protein with the same amino acid composition and sequence as in Methanococcus but it was acetylated at the N terminus as in the case with the homologous protein of E. coli. The in vivo incorporation of the overexpressed protein into the E. coli ribosomes was not observed. The overexpressed M. vannielii protein MvaL12e was incorporated into halobacterial ribosomes, thereby displacing the corresponding halobacterial L12 protein. Intact 70 S ribosomes were reconstituted from halobacterial 50 S subunits carrying the MvaL12e protein. These ribosomes were as active as native halobacterial ribosomes in a poly(U) assay. On the other hand, our attempts to incorporate L12 proteins from Bacillus stearothermophilus and E. coli into halobacterial ribosomes were not successful. These results support the conclusion which is based on primary sequence and predicted secondary structure comparisons that there exist two distinct L12 protein families, namely the eubacterial L12 protein family and the eukaryotic/archaebacterial L12 protein family.     

Comparative studies of ribosomal proteins and their genes from Methanococcus vannielii and other organisms

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacterium Methanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from the rif operon, according to the genome organization in Escherichia coli, and to proteins L23 and L2, which have comparable locations, as in the Escherichia coli S10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such as Halobacterium marismortui, Halobacterium halobium, or Sulfolobus. In general, the M. vannielii protein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of the Escherichia coli genome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine-Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.     

Structure-function relationships in the GTP binding domain of EF-Tu: mutation of Val20, the residue homologous to position 12 in p21.

Val20 of elongation factor Tu (EF-Tu), one of the best-characterized GTP binding proteins, is a variable residue within the consensus motif G-X-X-X-X-G-K involved in the interaction with the phosphates of GDP/GTP. To investigate the structure-function relationships of EF-Tu, which is widely used as a model protein, Val20 has been substituted by Gly using oligonucleotide-directed mutagenesis. The most important effects are: (i) a strong reduction of the intrinsic GTPase activity, (ii) a remarkable enhancement of the association and dissociation rates of EF-TuGly20-GDP, mimicking the effect of elongation factor Ts (EF-Ts) and (iii) the inability of ribosomes to influence the intrinsic GTPase of EF-Tu uncoupled from poly(Phe) synthesis. EF-TuGly20 can sustain poly(Phe) synthesis, albeit at a much lower rate than wild-type EF-TuVal20. As with the latter, poly(Phe) synthesis by EF-TuGly20 is inhibited by the antibiotic kirromycin, but differs remarkably in that it is largely independent of the presence of EF-Ts. According to primary sequence alignment, position 20 is homologous to position 12 of ras protein p21. As in p21, this position in EF-Tu is critical, influencing specifically the GDP/GTP interaction as well as other functions. The effect of the mutation displays diversities but also similarities with the situation reported for p21 having the corresponding residues in position 12. The differences observed with two homologous residues, Gly20 and Gly12 in EF-Tu and p21 respectively, show the importance of a variable residue in a consensus element in defining specific functions of GTP binding proteins.     

Gene for the diphtheria toxin-susceptible elongation factor 2 from Methanococcus vannielii.

Protein synthesis elongation factor 2 (EF-2) from all archaebacteria so far analysed, is susceptible to inactivation by diphtheria toxin, a property which it shares with EF-2 from the eukaryotic 8OS translation system. To resolve the structural basis of diphtheria toxin susceptibility, the structural gene for the EF-2 from an archaebacterium, Methanococcus vannielii, was cloned and its nucleotide sequence determined. It was found that (i) this gene is closely linked to that coding for elongation factor 1 alpha-(EF-1 alpha), (ii) the size of the gene product, as derived from the nucleotide sequence, lies between those for EF-2 from eukaryotes and eubacteria, (iii) it displays a higher sequence similarity to eukaryotic EF-2 than to eubacterial homologues, and (iv) the histidine residue which is modified to diphthamide and then ADP-ribosylated by diphtheria toxin is present in a sequence context similar to that of eukaryotic EF-2 but it is not conserved in eubacterial EF-G. The EF-2 gene from Methanococcus is expressed in transformed Saccharomyces cerevisiae but is not ADP-ribosylated by diphtheria toxin. This indicates that the Saccharomyces enzyme system is unable to post-translationally convert the respective histidine residue from the Methanococcus EF-2 into diphthamide.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene

The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.     

Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA

Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form. The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl). EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP. Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule. The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation. This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein. To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with [14C]Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined. The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 [Jonák, J., Petersen, T. E., Clark, B. F. C. and Rychlík, I. (1982) FEBS Lett. 150, 485-488]. Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B. stearothermophilus which are essential for the interaction with aminoacyl-tRNA. This is clearly reminiscent of a similar situation in E. coli EF-Tu [Jonák, J., Petersen, T. E., Meloun, B. and Rychlík, I. (1984) Eur. J. Biochem. 144, 295-303]. Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.     

The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial sfr operon

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF-G) and elongation factor Tu (EF-Tu), and ribosomal proteins S7 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF-G is described. Significant amino acid homology is also noted when the amino-terminal sequence of chlamydial EF-G is compared with the cytoplasmic tetracycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline-resistant clinical isolates of C. trachomatis are discussed.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.