ABSTRACT
Antibodies raised against a trpE-L16 fusion protein expressed in Escherichia coli were used to examine immunological relatedness between Saccharomyces cerevisiae ribosomal protein L16 and ribosomal proteins from eubacteria, halobacteria, methanogens, eocytes, and other eukaryotes. Homologues of L16 also were identified by searches of sequence data bases. Among the bacterial proteins that are immunologically related and similar in sequence to L16 are ribosomal proteins that bind 5 S rRNA. L16 protein fused near its carboxyl terminus to E. coli beta-galactosidase could assemble into functional yeast 60 S ribosomal subunits. The RPL16A-lacZ gene fusion partially complemented the slow growth or lethality of mutants containing null alleles of one or both RPL16 genes, respectively. L16-beta-galactosidase fusion protein cosedimented with ribosomes and polyribosomes, and remained associated with high salt-washed ribosomes. Monoclonal antibodies against beta-galactosidase were used to map the location of L16-beta-galactosidase on the surface of the 60 S subunit by immunoelectron microscopy. L16 was localized near the top surface of the central protuberance, where the 60 S subunit potentially contacts the 40 S subunit. This is similar to the location of the bacterial homologues of L16 in 50 S ribosomal subunits.
Subject(s)
Ribosomal Proteins/analysis , Ribosomes/chemistry , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Antibodies, Monoclonal , Bacteria/metabolism , Intracellular Membranes/chemistry , Intracellular Membranes/ultrastructure , Microscopy, Immunoelectron , Molecular Sequence Data , RNA, Bacterial/metabolism , RNA, Fungal/metabolism , RNA, Ribosomal, 5S/metabolism , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Ribosomal Proteins/immunology , Ribosomes/ultrastructure , Saccharomyces cerevisiae/immunology , beta-Galactosidase/immunology , beta-Galactosidase/metabolismABSTRACT
Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory. In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy. The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA. A novel structure, the platform ring, is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site. In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory. In order to evaluate the reliability of our model for the three-dimensional distribution of 16S rRNA, we have predicted which sites of rRNA are adjacent to ribosomal proteins and compared these predictions with r-protein protection studies of others. Good correlation between the model, the locations of rRNA sites, the locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins, provides independent support for this model.
Subject(s)
RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Bacterial Proteins/chemistry , Base Sequence , Escherichia coli/chemistry , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/chemistryABSTRACT
Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase.
Subject(s)
Bacillus subtilis/enzymology , Endopeptidases/analysis , Animals , Bacillus subtilis/physiology , Chloramphenicol/pharmacology , Culture Media , Endopeptidases/immunology , Enzyme Activation , Protease Inhibitors , Rabbits , Serine Endopeptidases , Spores, BacterialABSTRACT
The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.
