Crystallization of ribosomes from Thermus thermophilus
An understanding of the molecular bases of the process of protein biosynthesis on the ribosome requires a knowledge of its structure with high three-dimensional resolution involving the method of x-ray crystallographic analysis. The authors report on the production of crystals of the 70S ribosomes from a new source - the highly thermophilic bacterium Thermus thermophilus. Ribosomes for crystallization were obtained from Th. thermophilus strain HB8 by two washings in buffer with high ionic strength. The ribosome preparation was investigated for homogeneity by the method of high-speed sedimentation in a buffer containing 15 mM MgCl/sub 2/, 50 mM NH/sub 4/Cl, and 10 MM Tris-HCl, pH 7.5. Analysis showed that the preparation if homogeneous. The same preparation was investigated for intactness of ribosomal RNA by the method of gel electrophoresis in 2.75% acrylamide 0.5% agarose gel in a buffer containing 30 mM Tris, 30 mM NaH/sub 2/PO/sub 4/, 10 mM EDTA, 1-2% SDS, and 6 M urea. Analysis showed that the preparation possesses intact 16S and 23S RNA. The latter did not degrade, at least in a week of exposure of the ribosomes in buffer solution at 5/sup 0/C. The ribosome preparation had no appreciable RNase activity, which was verified by incubating 4.5 micrograms of ribosomes with 3 micrograms of /sup 14/C-labeled 16S rRna (50/sup 0/C, 90 min) in a buffer containing 10 mM MgCl/sub 2/, 100 mM NH/sub 4/Cl, and 10 mM Tris-HCl, pH/sub 20/sup 0// 7.5. The incubated nonhydrolyzed RNA was precipitated with 5% trichloroacetic acid and applied on a GF/C filter. The radioactivity was determined in a toluene scintillator on an LS-100C counter.
- Research Organization:
- Protein Institute, Pushchino, USSR
- OSTI ID:
- 5841916
- Journal Information:
- Dokl. Biochem. (Engl. Transl.); (United States), Vol. 289:1-6; Other Information: Translated from Dokl. Akad. Nauk SSSR; 289: No.5, 1263-1266(Aug 1986)
- Country of Publication:
- United States
- Language:
- English
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PROTEINS
BIOSYNTHESIS
RIBOSOMES
CRYSTALLIZATION
TRACER TECHNIQUES
RNA
LABELLING
BACTERIA
CARBON 14 COMPOUNDS
CYTOCHEMISTRY
ELECTRON MICROSCOPY
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ESCHERICHIA COLI
PRECIPITATION
RADIOACTIVITY
RNA-ASE
SCINTILLATION COUNTING
BIOCHEMISTRY
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
COUNTING TECHNIQUES
DECOMPOSITION
ENZYMES
ESTERASES
HYDROLASES
HYDROLYSIS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYSIS
MICROORGANISMS
MICROSCOPY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
PHASE TRANSFORMATIONS
PHOSPHODIESTERASES
SEPARATION PROCESSES
SOLVOLYSIS
SYNTHESIS
550301* - Cytology- Tracer Techniques
550201 - Biochemistry- Tracer Techniques