Piero Cammarano

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

Early evolutionary relationships among known life forms inferred from elongation factor EF-2/EF-G sequences: Phylogenetic coherence and structure of the archaeal domain

SummaryPhylogenies were inferred from both the gene and the protein sequences of the translational elongation factor termed EF-2 (for Archaea and Eukarya) and EF-G (for Bacteria). All treeing methods used (distance-matrix, maximum likelihood, and parsimony), including evolutionary parsimony, support the archaeal tree and disprove the “eocyte tree” (i.e., the polyphyly and paraphyly of the Archaea). Distance-matrix trees derived from both the amino acid and the DNA sequence alignments (first and second codon positions) showed the Archaea to be a monophyletia-holophyletic grouping whose deepest bifurcation divides a Sulfolobus branch from a branch comprising Methanococcus, Halobacterium, and Thermoplasma. Bootstrapped distance-matrix treeing confirmed the monophyly-holophyly of Archaea in 100% of the samples and supported the bifurcation of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 97% of the samples. Similar phylogenies were inferred by maximum likelihood and by maximum (protein and DNA) parsimony. DNA parsimony trees essentially identical to those inferred from first and second codon positions were derived from alternative DNA data sets comprising either the first or the second position of each codon. Bootstrapped DNA parsimony supported the monophyly-holophyly of Archaea in 100% of the bootstrap samples and confirmed the division of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 93% of the bootstrap samples. Distance-matrix and maximum likelihood treeing under the constraint that branch lengths must be consistent with a molecular clock placed the root of the universal tree between the Bacteria and the bifurcation of Archaea and Eukarya. The results support the division of Archaea into the kingdoms Crenarchaeota (corresponding to the Sulfolobus branch and Euryarchaeota). This division was not confirmed by evolutionary parsimony, which identified Halobacterium rather than Sulfolobus as the deepest offspring within the Archaea.

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1α (EF-1α) from the ultrathermophilic archaeotePyrococcus woesei: Phylogenetic implications

SummaryThe gene encoding elongation factor 1α (EF-1α, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeotePyrococcus woesei was localized within aBglII fragment of chromosomal DNA. Sequence analysis showed that the EF-1α gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNAser (GGA). The three genes follow each other immediately in the order EF-1α·S10·tRNAser after a putative promoter located 55 bp upstream of the EF-1α gene. Alignment of the derived EF-1α sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed thatPyrococcus EF-1α is highly homologous (89% identity) toThermococcus celer EF-1α, both being strikingly more similar to eukaryotic EF-1α than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony-although not evolutionary parsimony-support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of thePyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation ofPyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.

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

SummaryThe 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.

Arrangement and nucleotide sequence of the gene (fus) encoding elongation factor G (EF-G) from the hyperthermophilic bacterium Aquifex pyrophilus: Phylogenetic depth of hyperthermophilic bacteria inferred from analysis of the EF-G/fus sequences

The gene fus (for EF-G) of the hyperthermophilic bacterium Aquifex pyrophilus was cloned and sequenced. Unlike the other bacteria, which display the streptomycin-operon arrangement of EF genes (5′-rps12-rps7 fus-tuf-3′), the Aquifex fus gene (700 codons) is not preceded by the two small ribosomal subunit genes although it is still followed by a tuf gene (for EF-Tu). The opposite strand upstream from the EF-G coding locus revealed an open reading frame (ORF) encoding a polypeptide having 52.5% identity with an E. coli protein (the pdxJ gene product) involved in pyridoxine condensation. The Aquifex EF-G was aligned with available homologs representative of Deinococci, high G + C Gram positives, Proteobacteria, cyanobacteria, and several Archaea. Outgroup-rooted phylogenies were constructed from both the amino acid and the DNA sequences using first and second codon positions in the alignments except sites containing synonymous changes. Both datasets and alternative tree-making methods gave a consistent topology, with Aquifex and Thermotoga maritima (a hyperthermophile) as the first and the second deepest offshoots, respectively. However, the robustness of the inferred phylogenies is not impressive. The branching of Aquifex more deeply than Thennotoga and the branching of Thermotoga more deeply than the other taxa examined are given at bootstrap values between 65 and 70% in the fus-based phylogenies, while the EF-G(2)-based phylogenies do not provide a statistically significant level of support (⩽ 50% bootstrap confirmation) for the emergence of Thermotoga between Aquifex and the successive offshoot (Thermus genus). At present, therefore, the placement of Aquifex at the root of the bacterial tree, albeit reproducible, can be asserted only with reservation, while the emergence of Thermotoga between the Aquificales and the Deinococci remains (statistically) indeterminate.

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

SummaryThe glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the melting hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.

Secondary structure features of ribosomal RNA species of extremely thermoacidophilic archaebacteria (Caldariella acidophila), moderate thermoacidophilic and mesophilic eubacteria: Spectrophotometric studies☆

The L-rRNA and S-rRNA components of Caldariella acidophila (maximum growth temperature, 90°C), Bacillus acidocaldarius (maximum growth temperature, 70°C) and Escherichia coli (maximum growth temperature, 47°C), have been characterized by thermal ‘melting’ methods with respect to (i) heat stability, (ii) base composition of bihelical and single-stranded parts, (iii) length of the ‘independently melting’ double-helical segments, and (iv) helix content of the rRNA chains. (1) The rRNA species of C. acidophila are 11°C and 7°C higher in Tm than those of, respectively, E. coli and B. acidocaldarius. (2) Bihelical domains of the extreme thermophile rRNA (G·C%, 63–64) are richer in G·C basepairs than those of either B. acidocaldarius (G·C%, 59), or E. coli (G·C%, 55); the enhanced G·C content of the secondary structure structure regions is partly accounted for by a preferential concentration of G+C in bihelical parts, the single-stranded regions of the rRNA chains being 13–14% poorer in (G+C) than the base-paired regions. (3) The length of the ‘independently melting’ bihelical units has been calculated from the G·C content and Tm of the ‘melting’ species and found to be greater, on average, than those of both the mesophile and the moderate thermophile rRNA species; both the increased G·CA·U ratio and the increased average length of the ‘independently melting’ bihelical segments may contribute to the enhanced thermal stability of the C. acidophila rRNA species. (4) It is estimated by ‘melting’ hyperchromicity data that a fraction, 65–70% of the bases of the rRNA species investigated may be involved in hydrogen-bonded base-pairing, irrespective of degree of thermophily of the micro-organisms.

Immunochemical Cross-Reactivities of Protein Synthesis Elongation Factors (EF-Tu and EF-1α Proteins) Support the Phylogenetic Coherence of Archaebacteria

Summary Elongation factor Tu (EF-Tu) proteins have been purified by affinity chromatography on GDP-Sepharose columns, from the eubacterium Thermotoga maritima and from archaebacteria (Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum) representative of all known divisions in the arachaebacterial tree except halophiles. Polyclonal antibodies raised against the purified Tu proteins were challenged with homologous and heterologous factors including eukaryotic EF-1α and cross-reactivities were quantified using 125 I labelled Protein A as the reporter molecule. The immunochemical relationships among factors both within and across kingdom boundaries demonstrated that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the analogous proteins of eubacteria and eukaryotes, (ii) within the archaebacteria the immunochemical cross-reactivities grossly correlate with the phylogenetic relatedness of the organisms inferred from similarities in rRNA sequences. The data support the notion that archaebacteria form a phylogenetically coherent taxon.

Size heterogeneity of ribosomal RNA in eukaryote evolution—2. rRNA molecular weights in species containing discontinuous large ribosomal subunit RNA

1. The molecular weights and the integrity of the principal rRNA species derived from the large and small ribosomal subunit (respectively, L-rRNA and S-rRNA) of several species of Protostomia and Protozoa have been investigated. 2. Using gel electrophoresis in formamide, the molecular weights of protostomian L-rRNA species have been found to range from 1.30 X 10(6) (Annelida) to 1.61 X 10(6) (Diptera); those of the S-rRNAs cover the range 0.65 X 10(6) (Annelida)-0.81 X 10(6) (Diptera). 3. Both rRNA components have incurred extensive changes among the Protozoa; the L-rRNA ranges in weight from 1.35 X 10(6) (T. pyriformis) to 1.57 X 10(6) (A. castellanii) and the S-rRNA from 0.70 X 10(6) of T. pyriformis to 0.85 X 10(6) of A. castellanii and E. gracilis. 4. The L-rRNA components of all the species investigated are discontinuous molecules endowed with a latent median break; depending on whether the nick is located at the centre of the L-rRNA chain, or lies off-centre, the molecular weight of the S-rRNA equals that of either both, or only one, of the two fragments composing the L-rRNA.