Recognition mechanism of tRNA with tRNA(guanosine-2')methyltransferase from Thermus thermophilus HB 27

rRNA(Gm)methyltransferase from an extreme thermophile, Thermus thermophilus HB 27 specifically methylates the 2'-OH of the ribose ring of G18 in the invariant G18-G19 sequence in the D loop of tRNA. The interaction site on tRNA was presumed to be the D loop and stem structure. Destruction of tertiary structure of tRNA caused by heat resulted in a great decrease in the acceptor activity of methyl group. It was suggested by CD measurement that a conformational change of tRNA occurs when it forms an equimolar complex with Gm-methylase.     

Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.     

Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27

Recognition sites of tRNA by tRNA(guanosine-2'-)-methyltransferase (Gm-methylase) [EC 2.1.1.34] from an extreme thermophile, Thermus thermophilus HB27, were studied by two independent methods--fragment reactions and footprinting analyses, using yeast tRNA(Phe) and Escherichia coli tRNA(fMet) as substrates. None of the tRNA-derived oligonucleotides which have the G-G sequence but are not long enough to form the "stem-loop" structure could be methylated by Gm-methylase. The 5'-half fragments having the intact D-"stem-loop" structure served as substrates for Gm-methylase, with a similar Vmax but 6-8 times larger Km, as compared with the intact tRNAs. The results of footprinting analyses were consistent with the foregoing findings. Gm-methylase protected only the D-loop region of tRNA from RNase T1 attack, but other parts of tRNA extending from the amino acid stem to the T arm became more sensitive to RNase T1, suggesting a considerable change of tRNA tertiary structure due to complex formation with Gm-methylase. These results indicate that a D-"stem-loop" structure is a prerequisite for recognition by Gm-methylase.     

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNA^Phe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNA^Phe/(U)₉/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).     

tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8

tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8. The specific activity of the enzyme is about 50 000 and the yield of activity more than 20%. The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme. The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000. By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000. These data allow to suggest a subunit structure of the enzyme. The enzyme is highly thermostable and is most active at 80 degrees C. The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.     

Identification of Thermus thermophilus HB8 tRNA (Gm18) methyltransferase gene.

For the purpose of identification of the gene for Thermus thermophilus tRNA (Gm18) methyltransferase [tRNA (guanosine-2'-)-methyltransferase, EC 2.1.1.34], the purified enzyme from native source was analyzed by the peptide-mass mapping. The target gene encoded the amino acid sequences of the obtained peptides was searched in data from Thermus thermophilus HB8 genome-sequencing project. We found the target gene AB05130, which was expected to encode a protein composed of 194 amino acid residues and the molecular mass of this protein was calculated as 22083. The recombinant protein was expressed in E. coli as an active form. The Gm18 formation activity of the purified recombinant protein was confirmed by in vitro methylation followed by two-dimensional thin layer chromatography and Liquid Chromatography/Mass Spectrum analysis of substrate tRNA.     

Crystal structure of Thermus thermophilus tRNA m1A58 methyltransferase and biophysical characterization of its interaction with tRNA

Methyltransferases from the m¹A₅₈ tRNA methyltransferase (TrmI) family catalyze the S-adenosyl-l-methionine-dependent N₁-methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus TrmI, in complex with S-adenosyl-l-homocysteine, was determined at 1.7 Å resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI, and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis, and molecular docking. First, we confirmed the tetrameric oligomerization state of TrmI, and we showed that this protein remains tetrameric upon tRNA binding, with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Second, three key residues for the methylation reaction were identified: the universally conserved D170 and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N₉-methyladenine in the active site of TrmI. The N₉-methyladenine snugly fits into the catalytic cleft, where the side chain of D170 acts as a bidentate ligand binding the amino moiety of S-adenosyl-l-methionine and the exocyclic amino group of the adenosine. Y194 interacts with the N₉-methyladenine ring, whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78, and Y194) are essential for fashioning an optimized shape of the catalytic pocket.     

Thiostrepton-resistant mutants of Thermus thermophilus

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.     

Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8.

The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.     

Growth kinetics of Thermus thermophilus

Summary The nonsporulating extreme thermophile Thermus thermophilus was grown in continuous culture at dilution rates up to 2.65 h^–1 at 75°C and pH 6.9 on complex medium. Concomitantly very low yield (Y=0.12 g cell dry weight g^–1 utilized organic carbon) and incomplete substrate utilization (always less than 45%) were found. In batch cultures T. thermophilus could be grown with _max =h^–1, in shake flasks only with _max =h^–1 with the same low yield and incomplete substrate utilization. Stable steady states at 84C and 45°C were realized at a dilution rate of 0.3 h^–1 whereas at 86°C and 40°C no growth could be detected. Artefacts arising from wall growth (in bioreactors) or improper materials must be ruled out. Inhibition of growth by organic substrates was demonstrated at low concentrations: a decrease in the yield obtained was found when more than 0.7 gl^–1 of meat extract were supplied in the medium. The maintenance requirement for oxygen is potentially very high and was determined to be 10 to 15 mmol g^–1 h^–1.     

Thermus thermophilus as biological model

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.     

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA(Phe).

Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA(Phe). It is shown that the 'hyperaminoacylated' tRNA(Phe) is the bis-2',3'-O-phenylalanyl-tRNA(Phe), and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl-tRNA synthetase under the same conditions.     

Overproduction of carotenoids in Thermus thermophilus

Phytoene synthase encoded by the crtB gene is one of the rate-limiting enzymes for carotenoid production in Thermus thermophilus. We introduced a multicopy recombinant plasmid, pCOP1, in which the Thermus crtB gene was cloned, into carotenoid overproducing mutants of T. thermophilus. The overproducing mutants carrying a pCOP1 produced about twenty times as much carotenoids as the parental strain did.     

Transferable Denitrification Capability of Thermus thermophilus

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N₂O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.     

Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.