Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.
Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000378:metabolism / Q000648:ultrastructure*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010445:Peptide Elongation Factors; D010458:Peptidyl Transferases / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000032:analysis; D012269:Ribosomal Proteins / Q000032:analysis; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*
D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D010458:Peptidyl Transferases; D010727:Phosphoric Diester Hydrolases