ABSTRACT
The gene for the ribosomal protein S19 from Thermus thermophilus was cloned, sequenced and overexpressed in Escherichia coli. A simple procedure for isolating the recombinant protein was developed. Preliminary NMR studies revealed a high content of alpha-helical secondary structure in the protein.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Gene Expression , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Ribosomal Proteins/genetics , Sequence Alignment , Sequence Analysis, DNA , Thermus thermophilus/chemistryABSTRACT
The gene encoding the 5S rRNA-binding ribosomal protein TL5 from Thermus thermophilus, an extremely thermophilic species, was expressed in E. coli. A method for isolation of TL5 from the overproducing strain was developed. Samples of TL5 protein isolated from ribosomes and the overproducing strain displayed identical RNA-binding properties. Circular dichroic spectroscopy was used to calculate the secondary structure of the protein. TL5 was shown to form a stable complex with the 3'-terminal fragment of 5S rRNA, which is similar to the fragment of E. coli RNA that binds to L25 protein. The data suggest that TL5 from T. thermophilus and L25 from E. coli bind to similar sites on the 5S rRNA molecule.
Subject(s)
Bacterial Proteins , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Thermus thermophilus/metabolism , Binding Sites , Circular Dichroism , Escherichia coli/genetics , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Proteins/geneticsABSTRACT
The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced. The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria. The deduced amino acid sequence consists of 206 residues. The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC. The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli. An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved. No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA. The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.
Subject(s)
RNA-Binding Proteins/biosynthesis , Ribosomal Proteins/biosynthesis , Thermus thermophilus/metabolism , Amino Acid Sequence , Bacillus subtilis/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Base Sequence , Codon , Escherichia coli/metabolism , Genes, Bacterial , Molecular Sequence Data , RNA, Ribosomal, 5S/metabolism , RNA-Binding Proteins/chemistry , Ribosomal Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The gene for the ribosomal protein L22 from Thermus thermophilus has been sequenced and overexpressed in Escherichia coli. A multiple sequence alignment was carried out for all proteins of the L22 family reported so far. The recombinant protein was purified and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with cell parameters of a = 32.6 A, b = 66.0 A, c = 67.8 A.
Subject(s)
Escherichia coli Proteins , Genes, Bacterial/genetics , RNA-Binding Proteins/genetics , Ribosomal Proteins , Thermus thermophilus/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Molecular Sequence Data , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.
Subject(s)
Escherichia coli/enzymology , Protein Structure, Tertiary , Tetrahydrofolate Dehydrogenase/chemistry , Amino Acid Sequence , Base Sequence , Circular Dichroism , Enzyme Stability , Escherichia coli/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Denaturation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Tetrahydrofolate Dehydrogenase/drug effects , Tetrahydrofolate Dehydrogenase/genetics , Urea/pharmacologyABSTRACT
Using restriction enzymes and polymerase chain reaction, three mutated forms of L7/L12 proteins with deleted 38-46, 44-52 and 38-52 residues were obtained. These mutant proteins were isolated in a preparative scale and were shown to bind to ribosomes and to affect ribosomal function.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Chromosome Deletion , DNA/genetics , Escherichia coli , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , Ribosomal Proteins/genetics , Ribosomes/physiologyABSTRACT
The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.
