Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Duplication of the tuf gene, which encodes peptide chain elongation factor Tu, is widespread in Gram-negative bacteria.

The tuf gene which encodes peptide chain elongation factor Tu was found to be duplicated in nine enteric and four nonenteric gram-negative bacteria, but present only in one copy in two gram-positive genera. In two of the nonenteric gram-negative genera, Pseudomonas and Caulobacter, the duplicate tuf genes were found to be very close together on the chromosome, which contrasts with the situation in Escherichia coli, where they are more than 660 kilobases apart.     

The antibiotics kirromycin and pulvomycin bind to different sites on the elongation factor Tu from Escherichia coli

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichia coli by complex formation with the elongation factor Tu (EF-Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF-Tu, more than one molecule of pulvomycin interacts with a molecule of EF-Tu. This has been deduced from experiments in which the aminoacyl-tRNA binding and the GTPase activity of EF-Tu were measured in the presence of varying amounts of both antibiotics. These experiments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.     

Preparation of nucleotide-free elongation factor Tu and its stabilization by the antibiotic kirromycin

A rapid, highly reproducible procedure for the preparation of nucleotide-free elongation factor Tu (EF-Tu) is described. A microscale gel filtration is performed in a two-step elution, which takes less than 30 min and allows the preparation of nanomole amounts of the factor. The nucleotide-free EF-Tu is unstable, as measured by its ability to bind GDP. However, it can be stabilized either by the presence of a residual contamination of GDP of at least 1%, in the absence of Mg²⁺, or by kirromycin. In the presence of the latter component, the nucleotide-free EF-Tu is stable over a long period of time, similarly to the EF-Tu· GDP complex. Both GDP and kirromycin promote the reactivation of partially inactivated nucleotide-free EF-Tu, as measured by GDP binding and GTPase activity.     

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The binding of kirromycin to elongation factor Tu. Structural alterations are responsible for the inhibitory action.

The influence of kirromycin on the elongation factor Tu (EF-Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF-Tu . GDP and EF-Tu . GTP was determined by circular dichroism titrations to be 4 x 10(6) M-1, and to EF-Tu . GTP . aa-tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 x 10(6) M-1. In the presence of kirromycin the binding of aminoacyl-tRNAs to EF-Tu . GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl-tRNA is more accessible to the non-enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.     

Protein synthesis elongation factors Tu and Tu.Ts from Caulobacter crescentus: sensitivity to kirromycin and activity in Q beta replicase.

The protein synthesis elongation factors Tu and Ts are responsible for binding aminoacyl-transfer ribonucleic acid (RNA) to the ribosome. In addition, they perform an undefined function, as the EF-Tu.Ts complex, in the RNA phage RNA replicases. In an effort to obtain insight into these two apparently unrelated roles, we purified the elongation factors from Caulobacter crescentus and compared them to the analogous Escherichia coli polypeptides. Although most physical and functional characteristics were found to be similar, significant differences were found in the molecular weight of EF-Ts and relative affinities of guanine nucleotides, sensitivity to trypsin cleavage, and rate of heat denaturation of EF-Tu. The antibiotic kirromycin was active with EF-Tu from both bacterial species. When C. crescentus EF-Tu.Ts was substituted for the E. coli elongation factors in Q beta phage RNA replicase, an enzyme capable of apparently normal RNA synthetic activity was formed.     

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

Mutagenesis of the NH2-terminal domain of elongation factor Tu

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.     

The Neurospora crassa colonial temperature-sensitive 3 (cot-3) gene encodes protein elongation factor 2

At elevated temperatures, the Neurospora crassa mutant colonial, temperature-sensitive 3 (cot-3) forms compact, highly branched colonies. Growth of the cot-3 strain under these conditions also results in the loss of the lower molecular weight (LMW) isoform of the Ser/Thr protein kinase encoded by the unlinked cot-1 gene, whose function is also involved in hyphal elongation. The unique cot-3 gene has been cloned by complementation and shown to encode translation elongation factor 2 (EF-2). As expected for a gene with a general role in protein synthesis, cot-3 mRNA is abundantly expressed throughout all asexual phases of the N. crassa life cycle. The molecular basis of the cot-3 mutation was determined to be an ATT to AAT transversion, which causes an Ile to Asn substitution at residue 278. Treatment with fusidic acid (a specific inhibitor of EF-2) inhibits hyphal elongation and induces hyperbranching in a manner which mimics the cot-3 phenotype, and also leads to a decrease in the abundance of the LMW isoform of COT1. This supports our conclusion that the mutation in cot-3 which results in abnormal hyphal elongation/branching impairs EF-2 function and confirms that the abundance of a LMW isoform of COT1 kinase is dependent on the function of this general translation factor.