Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid

1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K(+) and 0.1m-Mg(2+), were extracted with low-ionic-strength buffer 75-80% of the 30S proteins and 60-65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li(+)-EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).     

Fractionation of native and reconstituted chromatin by digesting with deoxyribonuclease ii

Native and reconstituted chromatin from Ehrlich ascites tumor cells were fractionated into template-active and inactive fractions by the DNAase II/Mg2+-solubility method of Gottesfeld et al. (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F. and Bonner, J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). The Mg2+-soluble (template-active) fractions were compared in respect to sedimentation behavior in sucrose gradients and the relative content of specific transcribed (ribosomal) and non-transcribed (satellite) DNA sequences. It was found that the Mg2+-soluble fraction of the native chromatin was enriched in ribosomal DNA while almost completely devoid of satellite DNA; the nucleoprotein monomer of this fraction sedimented in sucrose gradient at 14 S. Similar-results were obtained if chromatin was fractionated in the presence of 3 M urea. With reconstituted chromatin, however, neither the sedimentation profile, nor the relative content of ribosomal and satellite DNA sequences were recovered, thus indicating that reconstitution did not yield nucleoprotein structurally similar to native chromatin.     

CHARACTERIZATION OF RAT BRAIN RIBONUCLEIC ACIDS BY AGAR GEL ELECTROPHORESIS

Abstract— —The characteristics of total and rapidly-labelled RNAs of rat brain were studied by agar gel electrophoresis. The bulk (more than 90 per cent) of total, nuclear and cytoplasmic brain RNA was represented by the 28 S, 18 S and 4 S RNA components. The 28 S/18 S RNA mass ratio in cytoplasmic RNA was 2·55. Lower values for this ratio were obtained with total and nuclear RNAs. Five minor RNA components were detected in total brain RNA with mobilities in agar gel corresponding to 24 S, 22 S, 14 S, 9 S and 6 S. Two broad rapidly labelled RNA components were detected in total and nuclear (but not in cytoplasmic) brain RNA with mobilities corresponding to about 45 S and 31 S. These fractions were of nuclear origin and resembled ribosomal precursor RNAs of other animal tissues. In cytoplasmic RNA the radioactivity and ultraviolet profiles coincided at all labelling times down to 1 hr. The G + C/A + U ratio of brain RNA was 1·50 for total RNA, 1·39 for nuclear RNA and 1·59 for cytoplasmic RNA. The G + C/A + U ratio of 1 hr-labelled total brain RNA (determined by ³²P-distribution) was 0·94. This ratio rose to 1·31 at 24 hr labelling. The possible significance of these results for the elucidation of ribosomal and messenger RNA metabolism in brain is discussed.     

Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.     

Identification of neighboring protein pairs in the 60 S ribosomal subunits from Saccharomyces cerevisiae by chemical cross-linking

Protein-protein cross-linking was used to determine the spatial arrangement of proteins within the 60 S ribosomal subunits of Saccharomyces cerevisiae. Protein cross-links were generated by treatment of intact ribosomal subunits with dimethyl 3,3'-dithiobispropionimidate. Proteins were extracted from the treated subunits and fractionated by Cm-cellulose chromatography. Cross-linked proteins in these fractions were analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing sodium dodecyl sulfate. Component members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Seventeen pairs involving 16 of the 45 60 S subunit proteins were identified. Several proteins were detected in numerous cross-linked dimers and were used as foci for constructing a model depicting the arrangement of proteins within the 60 S ribosomal subunit. The model also incorporated previously published data on structure and function of proteins from the yeast 60 S subunit.     

Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate. J. Bacteriol. 91:2139-2145. 1966.-Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 x g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 mug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10(-4)m MgCl(2) to the final diluent increased immunogenic activity, whereas larger concentrations (10(-3)m) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Two-week-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.     

Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)

Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in ³H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4–5 × 10⁵ dpm/μg. 4 + 5S, 18S and 25S RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18S and 25S RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.     

An unmethylated “3 SE” RNA in hamster mitochondria: A 5 S RNA-equivalent?

A novel low molecular weight (“3 S_E”) RNA associated with hamster cell mitochondria has been partially characterized. It was present at approx. 1:1 molar ratio with structural mitochondrial ribosomal RNA; it was unmethylated; and it resembled other mitochondrial RNA fractions in having a low content of G + C. These findings support the idea that 3 S_E RNA is a mitochondrial equivalent of 5 S ribosomal RNA.     

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.     

Ribosomes of Brachionus plicatilis (Rotifera) – distribution in homogenate fractions and general properties

(1) The content of DNA, RNA and of proteins of Brachionus plicatilis was estimated and the distribution of RNA and of proteins of different homogenate fractions characterised. (2) Ribosomes were isolated from Brachionus plicatilis homogenates and were characterised by gradient centrifugation. (3) Unlike the RNA content, the yield of ribosomes from different homogenate fractions is strongly dependent on the concentration of Mg²⁺-ions in the buffers. Likewise resuspension of ribosomes is more effective in Mg²⁺- (or Ca²⁺-) free buffers. (4) Dissociation of ribosomes was brought about by centrifugation of ribosomes in gradients containing less than 4 mM Mg²⁺. In this case, beside the peaks of subunits, a peak in the region of 80 S remained which vanished only under conditions destroying ribosomal material altogether. (5) Proteins were isolated from ribosomal subunits and from undissociated ribosomes and were characterised by two-dimensional gel electrophoresis techniques. Patterns of 51 spots were regularly obtained from large subunits and patterns of 41 spots from small subunits. The undissociated ribosomes showed 83 spots, most of which could be attributed to the large or the small subunit. The ribosomal proteins have molecular masses of between 11000 and 56000 Da, while the molecular mass of the total protein content of Brachionus ribosomes was estimated to be 1.8 ±0.5) ×10⁶ Da.     

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.     

Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000)

A ribosomal subunit antiassociation activity has been purified from both the postribosomal supernatant and ribosomal salt-wash protein fractions of rabbit reticulocyte lysates. A majority (greater than 90%) of the activity is associated with a low molecular weight protein of Mr of approximately 25,000. A small but significant level of antiassociation activity (less than 10%) was found to be associated with higher molecular weight protein fractions. The purified 25,000-dalton antiassociation factor interacts with 60 S ribosomal subunits to prevent them from reassociating with 40 S ribosomal subunits. The factor does not seem to interact directly with 40 S subunits nor does it dissociate 80 S monosomes. The properties of this factor are thus similar to the eukaryotic initiation factor 6 isolated from both wheat germ and calf liver extracts.     

Cross-linking study on protein topography of rat liver 60 S ribosomal subunits with 2-iminothiolane

Rat liver 60 S ribosomal subunits were modified with 2-iminothiolane. After treatment with hydrogen peroxide, the cross-linked proteins were extracted and then separated into 24 fractions by chromatography on carboxymethylcellulose. Each protein fraction was then analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis (Sommer, A., and Traut, R.R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3946-3950). The pieces of gel containing cross-linked protein spots that were shifted from the diagonal line were labeled with 125I. The labeled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. Fifty-three cross-linked protein pairs involving 35 protein species containing two acidic proteins were identified. From these and previous results, a preliminary model of the protein topography of the 60 S ribosomal subunit was constructed and discussed in relation to other functional data on 60 S ribosomal proteins.     

Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids.

Homogenates of rat liver obtained 3 or 14 days after partial hepatectomy were used to prepare the postmicrosomal pH5-supernatant fraction and to prepare salt-wash fractions of the 40S ribosomal subunits and the 80S ribosomes. The factor-dependent binding of methionyl-tRNAfMet to ribosomes and the elongation-factor-1-dependent binding of phenylalanyl-tRNA to ribosomes were both increased after 3 days of growth, but not after 14 days of growth. An activity inhibitory to phenylalanyl-tRNA binding that was located in ribosomal wash fractions was decreased after 14 days of growth. Since the decreased inhibitory activity was obtained from the ribosomes and was tested against ribosomes and excess of pH5-supernatant fraction from control rat liver, its action was separate from the phenylalanyl-tRNA binding activities of the pH5-supernatant fractions from sham-operated and regenerating liver.     

Structural change of ribosomes during apoptosis: degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, P0, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis.