Crystallization of ribosomes from Thermus thermophilus

Journal Article · Thu Jan 01 04:00:00 EST 1987 · Dokl. Biochem. (Engl. Transl.); (United States)

OSTI ID:5841916

Karpova, E A; Serdyuk, I N; Tarkhovskii, Yu S; Orlova, E V; Borovyagin, V L

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), Journal Name: Dokl. Biochem. (Engl. Transl.); (United States) Vol. 289:1-6; ISSN DBIOA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMISTRY
BIOSYNTHESIS
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
COUNTING TECHNIQUES
CRYSTALLIZATION
CYTOCHEMISTRY
DECOMPOSITION
ELECTRON MICROSCOPY
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
HYDROLYSIS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
LYSIS
MICROORGANISMS
MICROSCOPY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
PHASE TRANSFORMATIONS
PHOSPHODIESTERASES
PRECIPITATION
PROTEINS
RADIOACTIVITY
RIBOSOMES
RNA
RNA-ASE
SCINTILLATION COUNTING
SEPARATION PROCESSES
SOLVOLYSIS
SYNTHESIS
TRACER TECHNIQUES