The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an Mr of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.     

Proteins of the Bacillus stearothermophilus ribosome. The amino acid sequences of proteins S5 and L30

The amino acid sequences of ribosomal proteins S5 and L30 from Bacillus stearothermophilus have been determined. These proteins have recently been crystallized in our institute. Sequence data were obtained by manual sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin and chymotrypsin or thermolysin. Proteins S5 and L30 contain 166 and 62 amino acid residues and have calculated Mr values of 17,628 and 7,053, respectively. Comparison of the sequences with those of the homologous proteins from Escherichia coli shows 55% identical residues for S5 and 53% for L30. For both proteins, the distribution of conserved and substituted regions is not uniform throughout the molecule. Secondary structure predictions were carried out for the B. stearothermophilus proteins. Comparison with the results for the homologous E. coli proteins indicated similar secondary structural order for the molecules from the two species.     

The complete amino acid sequences of ribosomal proteins L17, L27, and S9 from Bacillus stearothermophilus

The complete primary structures of proteins L17, L27 and S9 extracted from the Bacillus stearothermophilus ribosomes with 1 M NaCl and purified to homogeneity by column chromatography have been determined. The amino acid sequences of these proteins are compared to those of the homologous ribosomal proteins from Escherichia coli. The number of identical amino acid residues between the homologous proteins lies between 33-55%.     

Proteins of the Bacillus stearothermophilus ribosome. Crystallization of proteins L30 and S5

Proteins L30 and S5 from the 50 S and 30 S subunits, respectively, of the Bacillus stearothermophilus ribosome have been crystallized. L30 crystals are tetragonal and the space group is P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 46.3 A and c = 61.4 A. S5 crystals are trigonal with the space group P3(1)21 (or P3(2)21) and cell dimensions a = b = 59.3 A and c = 109.8 A. In both cases, there appears to be a single molecule in the asymmetric unit.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.     

The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.     

The amino acid sequence of two small ribosomal proteins from Bacillus stearothermophilus

The low-Mr proteins (tentatively called protein I and II) were purified from 2 M NaCl extracts of the Bacillus stearothermophilus ribosome. Their amino acid sequences have been determined from the peptides obtained by digestion with trypsin, chymotrypsin, and pepsin, and by cleavage with CNBr, using the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205-214]. Protein I contains 56 residues and has an Mr of 6514. Protein II had 37 residues with an Mr of 4361. The amino acid sequence of protein I shows significant similarity to L32 from E. coli, whereas that of protein II is slightly, if at all, related to ribosomal protein L34 from E. coli.     

The Thermus thermophilus 5S rRNA-protein complex: Identifications of specific binding sites for proteins L5 and L18 in 5S rRNA

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilus have earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5-RNA complex crystallized, and its structure determined to 2.3 A. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment-L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.     

The crystallization of ribosomal proteins from the 50 S subunit of the Escherichia coli and Bacillus stearothermophilus ribosome

Several individual intact ribosomal proteins purified from bacterial sources under mild conditions have been crystallized. A number of these are suitable candidates for three-dimensional structural studies by x-ray diffraction techniques. Data collection to 3 A resolution for one of these proteins is in progress.     

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.     

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.     

The solution structure of ribosomal protein L18 from Bacillus stearothermophilus

A medium resolution solution structure has been obtained for L18 from Bacillus stearothermophilus (BstL18), a ribosomal protein that stabilizes the tertiary structure of 5S rRNA and mediates its interaction with the rest of the large subunit. The N-terminal 22 amino acid residues of BstL18 are unstructured in solution. Its remaining 98 residues form a globular domain that has the same topology as the globular domains of other L18s, but the orientation of helices is different. This conformational peculiarity should not prevent BstL18 from functioning in the ribosome the same way as other L18s.     

Proteins of the Bacillus stearothermophilus ribosome. A 5 A structure analysis of protein S5

The structure of protein S5 from the small subunit of the Bacillus stearothermophilus ribosome is described to a resolution of 5 A. The molecular boundary is visible and shows the protein to be essentially compact although slightly elongated in one dimension.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

5S rRNA binding proteins from the hyperthermophilic archaeon, Pyrococcus furiosus

A determination was made of the nucleotide sequence of the 2719 bp region of a ribosomal protein gene cluster (PfeL32-PfeL19-PfL18-PfS5-PfL30) containing a 5S rRNA binding protein L18 homolog of hyperthermophilic archaea Pyrococcus furiosus. The organization of the archaeal ribosomal protein gene cluster is similar to that in the spc-operon of Escherichia coli (L6-L18-S5-L30-L15) but has two additional genes, namely those encoding PfeL32 and PfeL19, which were identified as extra proteins that are apparently not present in bacterial E. coli. Using an inducible expression system, P. furiosus mature PfL18 protein and a mutant PfL18 with the basic N-terminal amino acid region deleted were produced in large amounts in E. coli and Northwestern analysis showed the N-terminal region of PfL18, including the conserved arginine-rich region, to have a significant role in 5S rRNA-PfL18 interaction.     

Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site.

A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA. It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent. No smaller resistant fragments were produced. 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association. Under milder trypsin digestion conditions three resistant fragments were produced from the free protein. The largest corresponded to that isolated from the complex. The smaller ones were trimmed slightly further at both N- and C-terminal ends. These smaller fragments did not reassociate with 5S RNA. It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction. The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA.     

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.     

Order of binding of substrate to valyl-tRNA synthetase from Bacillus stearothermophilus in amino acid activation reaction

Amino acid activation reaction with valyl-tRNA synthetase (EC 6.1.1.9) from Bacillus stearothermophilus was studied kinetically by measuring ATP-PPi exchange to find the order of the binding of substrate to the enzyme. The effects of the concentration of the substrates (L-valine and ATP) and two dead-end inhibitors (L-valinol and adenosine) on the reaction rate were analyzed. The results indicate that L-valine and ATP are bound to the enzyme in a random sequence. This conclusion is consistent with the one previously suggested by static binding experiments.     

The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1. The total intracellular concentrations of Na(+), K(+), Mg(2+), spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4-60 degrees . 5. Optimum binding occurred at about pH7.0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0.10-0.13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg(2+) on equal terms for sites on the ribosomes.