The complete primary structure of ribosomal protein L1 fromThermus thermophilus

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit ofThermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins fromEscherichia coli andBacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 fromT. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.     

The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon

To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T. thermophilus S8 protein as hybridization probe. The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined. Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria. However, T. thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E. coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E. coli) overlap. Most genes, except the rps14-rps8 intergenic spacer (69 bp), are separated by very short (only 3–7 bp) spacer regions or partially overlapped. The deduced aa sequences of T. thermophilus proteins share about 51–100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27–70% identities with the sequences of their mesophile counterparts.     

Domain II of Thermus thermophilus ribosomal protein L1 hinders recognition of its mRNA

The two-domain ribosomal protein L1 has a dual function as a primary rRNA-binding ribosomal protein and as a translational repressor that binds its own mRNA. Here, we report the crystal structure of a complex between the isolated domain I of L1 from the bacterium Thermus thermophilus and a specific mRNA fragment from Methanoccocus vannielii. In parallel, we report kinetic characteristics measured for complexes formed by intact TthL1 and its domain I with the specific mRNA fragment. Although, there is a close similarity between the RNA-protein contact regions in both complexes, the association rate constant is higher in the case of the complex formed by the isolated domain I. This finding demonstrates that domain II hinders mRNA recognition by the intact TthL1.     

Proteins of the Thermus thermophilus ribosome Purification of several individual proteins and crystallization of protein TL7

The procedure of selective removal of eight proteins from the 50 S ribosomal subunit of the extreme thermophilic bacterium Thermus thermophilus has been developed based on extraction at 60°C in the presence of 0.5 M or 1 M NH₄Cl and 50% ethanol. CM-Sepharose CL column chromatography of the protein mixture under non-denaturing conditions yielded five proteins with a purity of 95% or higher. Crystals of one of these proteins, namely TL7 (probably an analog of L6 protein from the Escherichia coli ribosome) have been obtained using the ‘hanging drop’ method with ammonium sulphate as a precipitant.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

Identification of Ribosomal Protein L1-Binding Sites in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis> mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.     

Crystal structures of mutant ribosomal proteins L1

Nine mutant ribosomal proteins L1 from the bacterium Thermus thermophilus and archaeon Methanococcus jannaschii were obtained and their crystal structures were determined and analyzed. The structure of the S179C TthL1 mutant, determined earlier, was also analyzed. In half of the proteins studied, point mutations of the amino acid residues exposed on the protein surface essentially changed the spatial structure of the protein. This proves that a correct study of biological processes with the help of site-directed mutagenesis requires a preliminary determination or, at least, modeling of the structures of mutant proteins. A detailed comparison of the structures of the L1 mutants and the corresponding wild-type L1 proteins demonstrated that the side chain of a mutated amino acid residue tends to adopt a location similar to that of the side chain of the corresponding residue in the wild-type protein. This observation assists in modeling the structure of mutant proteins.     

Domain I of ribosomal protein L1 is sufficient for specific RNA binding

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

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.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

E. coli ribosomal protein L4 is a feedback regulatory protein

We studied the synthesis of ribosomal proteins encoded by the S10 operon, an eleven gene operon from the str-spc region of the E. coli chromosome, using a λfus3 DNA-directed, in vitro protein synthesizing system. Addition of ribosomal protein L4 (1 μM) to in vitro protein synthesis reactions caused selective inhibition of synthesis of the promoter-proximal proteins of the S10 operon, S10, L3, L4, L23 and possibly L2. Proteins of the S10 operon other than L4 did not cause selective inhibition of protein synthesis. Autoregulatory ribosomal proteins previously identified from other operons, L1, S4 and S8, did not inhibit protein synthesis from the S10 operon; nor did L4 cause significant inhibition of protein synthesis from operons other than the S10 operon. As with L1, S4 and S8, L4 inhibits gene expression at the level of translation.     

Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

A characterization by sequencing of the termini of the polypeptide chain of cyclic AMP receptor protein from Escherichia coli and the subtilisin produced N-terminal fragment

The complete primary structure of protein L2 which is the largest protein component of the E. coli 50 S subunit, has been established. A combination of enzymatic and chemical cleavages has been employed to isolate peptides, which were sequenced by the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205–214]. The sequence determined shows protein L2 to consist of 272 amino acid residues with M_r = 29730. Secondary structure predictions were made based on the primary structure. Further, sequence regions homologous to other ribosomal proteins are presented. These results suggest protein L2, which binds specifically to the 23 S RNA, to show homologous sequence stretches to the other RNA-binding proteins.     

Functional domains of Escherichia coli Ribosomal Protein S1. Formation and characterization of a fragment with ribosome-binding properties.

Treatment of Escherichia coli ribosomal protein S1 with TPCK-treated trypsin under mild conditions (0 °C, 1 to 2 μig trypsin/mg S1 protein) results in the production of a high molecular weight fragment in yields of up to 80% within a few minutes. The fragment is relatively resistant to further degradation. We have isolated the fragment in pure form for structural and functional characterization. The fragment (denoted S1-F1) has a molecular weight of 48,500 as shown by sodium dodecyl sulphate gel electrophoresis, and therefore it contains approximately 60% of the amino acid residues of S1. The N-terminal sequence of the fragment is different from that of intact S1.The fragment binds to the 30 S ribosomal subunit and to polyuridylic acid in approximately the same manner as intact S1, indicating that the active centres of S1 concerned with these two characteristic binding properties are localized within the fragment. In spite of the above properties, the fragment was completely unable to support protein synthesis. The significance of these results in relation to the structure and function of S1 is discussed.     

Spatial organization of catalase proteins

The three-dimensional structure of the heme-containing fungal catalase fromPenicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2.0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct “X-ray” primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains aβ-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near theβ-barrel. A comparison of the structure of catalase fromPenicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteriaThermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2₁3, a = 133.4 Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme inThermus thermophilus catalase, the active site is between the four helices and contains two manganese ions.     

Influence of Nonconserved Regions of L1 Protuberance of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> Ribosome on the Affinity of L1 Protein to 23s rRNA

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.