Ribosome specificity of archaebacterial elongation factor 2. Studies with hybrid polyphenylalanine synthesis systems

Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus. By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom. In contrast, EF-1 from T. thermophilus significantly cooperated with ribosomes from T. acidophilum.     

Studies on DNA polymerases and topoisomerases in archaebacteria

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.     

Tryptophan biosynthesis genes trpEGC in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

A DNA fragment containing the trpEGC gene cluster was isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The products of trpE, trpG, and trpC from S. solfataricus were compared to the homologous products from a eukaryote, a eubacterium, and two archaebacteria, namely, a methanogen and an extreme halophile. They appeared to be equally related to the proteins from Escherichia coli and Saccharomyces cerevisiae, the percentages of conserved amino acids being roughly the same as those measured when comparing the eubacterial and eukaryotic sequences directly. These percentages did not rise significantly when a comparison with the proteins from Haloferax volcanii was drawn, while a slightly closer relationship with the proteins from Methanococcus thermoautotrophicum was found.     

Unique antibiotic sensitivity of an in vitro polypeptide synthesis system from the archaebacterium Thermoplasma acidophilum. Phylogenetic implications

Summary The susceptibility of Thermoplasma acidophilum (an extremely acidophilic, moderately thermophilic, wall-less sulphur-oxidizing archaebacterium) to 50 ribosome-specific inhibitors of polypeptide elongation was surveyed using efficient poly(U)-and poly(UG)-directed cell-free systems and comparable reference systems derived from eubacterial (Bacillus stearothermophilus, Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) species. Under optimum temperature (58° C) and ionic conditions for polypeptide synthesis Thermoplasma ribosomes are only sensitive to the 70 S/80 S ribosome-directed aminoglycoside neomycin, and to five 80 S ribosome-directed inhibitors all of which (-sarcin, mitogillin, restrictocin, dianthin and gelonin) impair the functioning of the large (60 S) ribosomal subunit. Sensitivity of the three structurally related compounds -sarcin, mitogillin and restrictocin and susceptibility to neomycin place Thermoplasma ribosomes between those of Sulfolobus solfataricus (only sensitive to -sarcin) and Methanococcus vannielli (sensitive to -sarcin, mitogillin, restrictocin and neomycin but also affected by a variety of 70 S ribosome-directed drugs). The phylogenetic significance of the greatly diversified antibiotic sensitivity spectra displayed by archaebacteria in general, as opposed to the uniform ones exhibited by eubacteria and eukaryotes, is discussed.     

Sequence of the 16S rRNA gene from the thermoacidophilic archaebacteriumSulfolobus solfataricus and its evolutionary implications

Summary The sequence of the small-subunit rRNA from the thermoacidophilic archaebacteriumSulfolobus solfataricus has been determined and compared with its counterparts from halophilic and methanogenic archaebacteria, eukaryotes, and eubacteria. TheS. solfataricus sequence is specifically related to those of the other archaebacteria, to the exclusion of the eukaryotic and eubacterial sequences, when examined either by evolutionary distance matrix analyses or by the criterion of minimum change (maximum parsimony). The archaebacterial 16S rRNA sequences all conform to a common secondary structure, with theS. solfataricus structure containing a higher proportion of canonical base pairs and fewer helical irregularities than the rRNAs from the mesophilic archaebacteria.S. solfataricus is unusual in that its 16S rRNA-23S rRNA intergenic spacer lacks a tRNA gene.     

Some biochemical properties of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H⁺-ATPase inhibitors, these enzymes seemed not to be F₀F₁-ATPase.     

Inferring the phylogeny of archaebacteria: The use of ribosomal sensitivity to protein-synthesis inhibitors

Summary The sensitivities to 37 protein-synthesis inhibitors were determined in cell-free translational systems for 13 Archaebacteria species. Multivariate factor analysis by principal components, cluster analysis by the unweighted pair-group clustering algorithm, and compatibility and parsimony methods of constructing phylogenetic trees were used both to infer the genealogical relationships within archaebacteria and to establish their relations with the eubacterial and eukaryotic kingdoms.     

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A+C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial beta' and chloroplast beta' and beta" subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial beta' lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.     

CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the CCA-adding enzyme from the archaeal hyperthermophile Sulfolobus shibatae.

We describe the purification, cloning, and characterization of the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] from the thermophilic archaebacterium, Sulfolobus shibatae. Characterization of an archaeal CCA-adding enzyme provides formal proof that the CCA-adding activity is present in all three contemporary kingdoms. Antibodies raised against recombinant, expressed Sulfolobus CCA-adding enzyme reacted specifically with the 48-kDa protein and fully depleted all CCA-adding activity from S. shibatae crude extract. Thus, the cloned cca gene encodes the only CCA-adding activity in S. shibatae. Remarkably, the archaeal CCA-adding enzyme exhibits no strong homology to either the eubacterial or eukaryotic CCA-adding enzymes. Nonetheless, it does possess the active site signature G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][CLIVMFY]3-5 of the nucleotidyltransferase superfamily identified by Holm and Sander (1995, Trends Biochem Sci 20:345-347) and sequence comparisons show that all known CCA-adding enzymes and poly(A) polymerases are contained within this superfamily. Moreover, we propose that the superfamily can now be divided into two (and possibly three) subfamilies: class I, which contains the archaeal CCA-adding enzyme, eukaryotic poly(A) polymerases, and DNA polymerase beta; class II, which contains eubacterial and eukaryotic CCA-adding enzymes, and eubacterial poly(A) polymerases; and possibly a third class containing eubacterial polynucleotide phosphorylases. One implication of these data is that there may have been intraconversion of CCA-adding and poly(A) polymerase activities early in evolution.     

Immunological heterogeneity of archaebacterial protein synthesis elongation factors Tu (EF-Tu)

Abstract Polyclonal antibodies were raised against the EF-Tu of the archaebacterium Sulfolobus solfataricus and cross-reactivities of EF-Tus of other, phylogenetically disparate archaebacteria were determined using Western blotting and ELISA. The results demonstrate a high degree of heterogeneity of archaebacterial Tu factors with recognition by S. solfataricus EF-Tu antibodies ranging from 48% to 1.5% that observed with the homologous antigen. The immunochemical relatedness between the heterologous and the cognate ( Sulfolobus ) antigens correlates satisfactorily with similarities in 16 S rRNA sequences, there being no recognition of eubacterial and eukaryotic factors by the S. solfataricus EF-Tu antibodies.     

Archaebacterial and eukaryotic ribosomal subunits can form active hybrid ribosomes

Purified ribosomal subunits from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus are able to recognize ribosomal subunits from the yeast Saccharomyces cerevisiae forming hybrid monosomes that can be revealed by sucrose gradient analysis and are active in peptide bond formation. Both reciprocal combinations (archaebacterial 30 S + eukaryotic 60 S and archaebacterial 50 S + eukaryotic 40 S) are functional. In contrast, no hybrid couples are formed between subunits of yeast and Escherichia coli ribosomes. These results indicate that ribosomes of at least one archaebacterial species share specific structural features with those of the lower eukaryote S. cerevisiae.     

Aminoglycoside-induced mistranslation in thermophilic archaebacteria

Summary The effect of selected aminoglycoside antibiotics on the translational accuracy of poly(U) programmed ribosomes derived from the thermophilic archaebacteria Thermoplasma acidophilum, Sulfolobus solfataricus, Thermococcus celer and Desulfurococcus mobilis has been determined. Under optimum temperature and ionic conditions for polyphenylalanine synthesis, the four species investigated are found to be markedly diverse in their response to the miscoding-inducing action of aminoglycoside antibiotics. T. acidophilum is sensitive to all of the compounds tested except streptomycin; S. solfataricus responds to paromomycin and to hygromycin B; T. celer is only affected by neomycin, and D. mobilis is refractory to all drugs. The only feature shared by the four species under study, and by all archaebacteria so far investigated, is their complete insensitivity to streptomycin. The structural and phylogenetic implications of the remarkable diversity encountered among archaebacterial ribosomes in their susceptibility to aminoglycosides are discussed.     

A gene in the archaebacterium Sulfolobus solfataricus that codes for a protein equivalent to the alpha subunits of the signal recognition particle receptor in eukaryotes

We have sequenced a gene in the archaebacterium Sulfolobus solfataricus that codes for a protein that shows sequence similarity to the alpha subunit of the signal recognition particle receptor or docking protein in eukaryotes and the product of the ftsY gene in Escherichia coli. Comparison of the Sulfolobus 'docking protein' with its eukaryotic and eubacterial counterparts showed that the region of highest sequence similarity corresponds to a GTP-binding site. The presence of this gene in archaebacteria suggests that some of the components involved in protein transport have been conserved in the three kingdoms.     

Early assembly proteins of the large ribosomal subunit of the thermophilic archaebacterium Sulfolobus. Identification and binding to heterologous rRNA species

Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent. In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus. The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes. Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified. Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes. By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated.     

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.     

Archaebacterial malate dehydrogenases. The enzymes from the thermoacidophilic organisms Sulfolobus acidocaldarius and Thermoplasma acidophilum show A-side stereospecificity for NAD+.

The stereoselective transfer of hydrogen from NADH to oxaloacetate catalysed by malate dehydrogenases (EC 1.1.1.37) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius and Thermoplasma acidophilum was studied by the p.m.r. method described by Zhou & Wong [(1981) J. Biochem. Biophys. Methods 4, 329-338]. Both enzymes are A-side (pro-R) stereospecific for NADH.     

Action of synergimycins and macrolides on in vivo and in vitro protein synthesis in archaebacteria

Summary Single synergimycins (virginiamycin M or VM, type A component, and virginiamycin S or VS, type B component) inhibit reversibly growth and protein synthesis in Bacillus subtilis; a mixture of VM and VS produces viability loss and irreversible halting of peptide bond formation. In vitro, VM produces a five- to tenfold increase of the affinity of Escherichia coli ribosomes for VS, and erythromycin, which competes with VS for binding to eubacterial 50S subunits, is ineffective in the presence of VM. In the present work, the action of synergimycins and macrolides has been explored in vivo and in vitro on methanogenic and sulphurdependent archaebacteria. Multiplication of Methanococcus vannielii was synergistically inhibited by VM plus VS (for technical reasons, the action of synergimycins on growth and viability of most archaebacteria was unverifiable). When assayed on cell-free systems for protein synthesis from methanogens, both macrolides and single synergimycins were found ineffective. However, a mixture of VM and VS strongly inhibited poly(U)-directed polyphenylalanine synthesis. Binding of erythromycin to archaebacterial ribosomes and subunits was 10% (Mc. vannielii) or less than the control value (E. coli), and was not competed for by tylosin. The association constant of VS-50S complex formation, although low in the case of Mc. vannielii (as compared to enterobacteria), underwent a 100-fold increase in the presence of VM and was unaffected by macrolides. These data further stress the difference of the organization for protein synthesis of eubacteria and archaebacteria.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

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.