RESUMEN
The crystal structure of the mutant S179C of the ribosomal protein L1 from Thermus thermophilus has been determined at 1.9 A resolution. The mutant molecule displays a small but significant opening of the cavity between the two domains. The domain movement seems to be facilitated by the flexibility of at least two conserved glycines. These glycines may be necessary for the larger conformational change needed for an induced fit mechanism upon binding RNA. The domain movement makes a disulfide bridge possible between the incorporated cysteines in two monomers of the mutant L1.
Asunto(s)
Conformación Proteica , Proteínas Ribosómicas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Ribosómicas/genética , Thermus thermophilus/genéticaRESUMEN
L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ARN/química , Proteínas Ribosómicas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
A procedure for isolation in preparative amounts of 15 individual proteins from ribosomal 30S subparticles of Thermus thermophilus under non-denaturing conditions, has been developed. The amino acid composition and molecular masses of the proteins have been determined and the UV absorption spectra and extinction coefficients measured. A homology of 13 proteins to corresponding ribosomal proteins of E. coli has been established.
Asunto(s)
Proteínas Ribosómicas/aislamiento & purificación , Thermus thermophilus/química , Secuencia de Aminoácidos , Cromatografía en Gel , Escherichia coli/química , Datos de Secuencia Molecular , Peso Molecular , Desnaturalización Proteica , Proteínas Ribosómicas/química , Espectrofotometría UltravioletaRESUMEN
The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four-stranded anti-parallel beta-sheet with two alpha-helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA-interacting motif. Related topologies are also found in several other nucleic acid-interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA-interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid-interacting domain in large classes of ribonucleic acid binding proteins should be considered.
Asunto(s)
Proteínas Ribosómicas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Gráficos por Computador , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación Proteica , Proteína S6 Ribosómica , Homología de Secuencia de AminoácidoRESUMEN
The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit of Thermus 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 from Escherichia coli and Bacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 from T. 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.
Asunto(s)
Estructura Secundaria de Proteína , Proteínas Ribosómicas/química , Thermus thermophilus/metabolismo , Secuencia de Aminoácidos , Escherichia coli/metabolismo , Geobacillus stearothermophilus/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Ribosómicas/aislamiento & purificación , Homología de Secuencia de Aminoácido , TripsinaRESUMEN
Protein S8 from Thermus thermophilus consists of 138 amino acids of M(r) 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 from T. thermophilus shares a high percentage of identity with protein S8 from Thermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.
Asunto(s)
Proteínas Ribosómicas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Hidrólisis , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Thermus/químicaRESUMEN
In parallel with crystallographic studies of ribosomes from Thermus thermophilus, a long-term program on the crystallization and structural investigations of ribosomal proteins from the same microorganism has been started at the Institute of Protein Research (Pushchino, Russia). At present, more than half of the individual ribosomal proteins from T thermophilus have been purified without denaturating agents on a preparative scale and some of them have been obtained in the crystalline form. X-ray structural analysis of two ribosomal proteins, L1 and S6, is being carried out jointly with the Institute of Molecular Biology (Moscow, Russia) and laboratory of professor A Liljas (Lund University, Sweden). L1 is the large protein of the large ribosomal subunit. It can bind not only to a specific site on the 23S rRNA, but also to the mRNA that codes for L1 and L11, thereby acting as a translational repressor for the synthesis of these proteins. The crystals of L1 are orthorhombic and diffract to about 2 A resolution. Native data and data for several heavy atom derivatives have been collected. S6 is a small acidic protein from the small ribosomal subunit. The crystals of S6 are orthorhombic and diffract to 2 A resolution. Native data and derivatives' data have been collected.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Ribosómicas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Datos de Secuencia Molecular , Proteínas Ribosómicas/aislamiento & purificaciónRESUMEN
Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.
Asunto(s)
Proteínas Ribosómicas/química , Thermus/genética , Secuencia de Aminoácidos , Cristalización , Escherichia coli/genética , Datos de Secuencia Molecular , Proteína S6 Ribosómica , Proteínas Ribosómicas/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Difracción de Rayos XRESUMEN
Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.