Treatment Of Ribosomes Research Articles

In vivo analysis of the acidic ribosomal proteins BmP1 and BmP2 of the silkworm Bombyx mori in the yeast Saccharomyces cerevisiae

In the silkworm Bombyx mori the ribosomal stalk P-protein family consists of two low MW acidic proteins, BmP1 and BmP2, and of one higher MW protein, BmP0, as shown by electrophoretical and immunoblotting western blot analysis of purified ribosomes. Treatment of ribosomes with alkaline phosphatase followed by electrofocusing shifted the isoelectric points to higher pH, implying phosphorylation of the proteins. The cDNAs encoding BmP1 and BmP2 proteins were constructed and expressed in the Saccharomyces cerevisiae mutant strains defective in either the endogenous P1 or P2 proteins. The recombinant silkworm proteins could complement the absence of the homologous yeast proteins and were incorporated to the ribosomes of the transformed strains, helping the binding of the remaining endogenous acidic proteins, present in the cytoplasm in different extent. Thus, BmP1 was able to replace YP1α, preferentially binding YP2β to the ribosome, while BmP2 replaced both yeast P2 proteins and induced the binding of both YP1α and YP1β.

Ribosomes Deprived of Select Proteins Show Similar Structural Alterations Induced by Thermal Treatment of Native Particles

Three different techniques--light scattering, radiowave dielectric spectroscopy, and fluorescence were employed to investigate conformational variations in Escherichia coli ribosomes induced by removal of specific proteins. To this end, particles were treated with lithium chloride at different ion strength values to produce ribosomal cores. It was previously observed that treatment of ribosomes to subdenaturing temperatures promotes a structural rearrangement that implies a higher exposure of ribosomal RNA to the solvent. Results presented here strongly suggest that protein elimination from the ribosomal particle produces an overall response recalling the same variation of physical parameters previously observed after thermal treatment. We therefore suggest that high salt treatment produces the same structural modification caused by exposure to subdenaturing temperatures.

RIP-JIP60 Alters Conformation of Ribosomesin Vivo

Recently it has been demonstrated that expression putative ribosome-inactivating protein JIP 60 in transgenic tobacco plants leads to the depurination of large rRNA and inactivation of plant ribosomes. Treatment of ribosomes from transgenic tobacco plants with low concentration of α-sarcin resulted in the appearance of an rRNA specific fragment. No fragment was observed under the same conditions for ribosomes from wild type plants. The alteration of the comformation of ribosomes in transgenic tobacco due to the expression of JIP60 is assumed.

Mapping of nuclease-sensitive sites in native reticulocyte ribosomes--an analysis of the accessibility of ribosomal RNA to enzymatic cleavage.

Treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium-dependent nuclease from Staphylococcus aureus resulted in cleavage of rRNA. The positions of the cleaved phosphodiester bonds were localised by primer extension and polyacrylamide gel electrophoresis. S. aureus nuclease-induced strand scissions were found in the 5'-domain of 18S rRNA and in domains II, IV and VI of 28S rRNA. The majority of the cleavage sites were located in eukaryote-specific expansion segments and only one cleavage site was found in a region suggested to be directly involved in ribosomal function. Treatment of the reticulocyte lysate with increasing amounts of S. aureus nuclease resulted in the introduction of new cleavage sites. However, even at the highest nuclease concentration used, large parts of the rRNAs were protected from nuclease digestion. Removal of translational components, by salt wash of isolated reticulocyte polysomes, exposed additional rRNA sequences to S. aureus nuclease cleavage. These sequences were found in the 3'-major domain of 18S rRNA and in domains II, IV, and V of 28S rRNA. These sites are located at the putative translational surface of the ribosome. The translational activity of the S. aureus nuclease-treated ribosomes, determined after addition of exogenous mRNA, was directly correlated to the extent of nuclease digestion of the ribosomes. However, the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease was not due to a preferential exclusion of damaged ribosomes from polysome formation. This suggests that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the mRNA.

Acidic ribosomal proteins of Dictyostelium discoideum that show electrophoretic similarity to Escherichia coli proteins L7 and L12

This study documents the presence of three acidic proteins, A1 (pI 4.95), A2 (pI 4.85), and A3 (pI 4.70), in Dictyostelium discoideum ribosomes. All three proteins showed an apparent molecular mass of 13,000 by two-dimensional, sodium dodecyl sulfate gel electrophoresis. They were selectively released by treatment of ribosomes with 50% ethanol -1 M NH4Cl. The amino acid composition of A1, A2, and A3 were identical and indicated a predominant amount of alanine. All the above properties are shared by Escherichia coli proteins L7 and L12 and acidic ribosomal proteins in many eukaryotes. Unlike other eukaryotic systems, the acidic proteins of D. discoideum were found associated with the 40S rather than the 60S ribosomal subunit. Acidic proteins analogous in size and electrophoretic mobility to those of D. discoideum were also detected in several other cellular slime mold strains. Not one of the cellular slime mold acidic proteins reacted with antibodies to E. coli proteins L7 and L12 in immunodiffusion tests. In D. discoideum, the distribution of acidic proteins was altered during development. Amoebae contained all three proteins. In spores, A1 was absent and the relative amounts of A2 and A3 were lower than in amoebae. In addition, nine other acidic ribosomal proteins exhibited differences between vegetative amoebae and spores.

4-Dimethylaminoazobenzene interferes with the binding of ribosomes to reticular membranes

Treatment of chemically degranulated rough endoplasmic reticulum (RER) with 4-dimethylamino-azobenzene (4-DAB) partly damages binding of fresh polyribosomes to both the lighter and heavier fractions of reconstituted RER. Also, treatment of ribosomes with 4-DAB seemed to inhibit their binding to the chemically stripped RER (DRER) appearing both as lighter and heavier rough membrane layers after reconstitution. The interaction of 4-DAB with stripped microsomal fraction and ribosomes might be introducing certain conformational constraints into their proteins, thus crippling the binding of the ribosomes to reticular membranes.

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: The RNA N-glycosidase activity of the proteins

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.

Ribosomal protein phosphorylation induced during Q fever or by lipopolysaccharide: in vitro translation is stimulated by infected liver ribosomes

Q fever, as well as the lipopolysaccharide prepared from the rickettsial agent Coxiella burnetii, stimulates the phosphorylation of guinea pig liver ribosomal protein S6. In vitro mRNA and ribosome-dependent rabbit reticulocyte lysate translation systems reconstituted with ribosomes and mRNAs from infected animal livers were more active than those with mRNAs and ribosomes from uninfected animals. Treatment of ribosomes with a ribosomal supernatant phosphatase reduced the in vitro translation activities; the largest decreases occurred in systems with ribosomes and mRNAs from infected liver. These experiments provide a basis for explaining the increased hepatic protein synthesis during Q fever and demonstrate, perhaps for the first time, the phosphorylation of ribosomal protein in response to lipopolysaccharide. The implications of these observations are discussed in the context of previous studies on stimulated transcription and translation during Q fever.

Detection of antibodies against Candida albicans ribosomes by the enzyme linked immunosorbent assay.

Enzyme linked immunosorbent assay was found to be a convenient method for the investigation of antibodies in mice immunized with Candida albicans ribosomes. Antibodies against the ribosomal antigen were detected in all the sera of mice (ICR and BALB/c) immunized with ribosomes and incomplete Freund's adjuvant and in some of the sera of mice immunized with ribosomes only; the titer of antibodies varied from 1:320 to 1:10 240. Vaccination of mice with ribosomal protein and IFA resulted in a high titer of antiribosomal antibodies. Treatment of ribosomes with pronase abrogated the capacity of the ribosomes to elicit anti ribosomal humoral responses, suggesting that the antibodies detected were directed against the protein moiety of the ribosomes. The presence of antibodies in sera of immunized mice could not be correlated with the protection afforded by the ribosomal vaccination.

Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.

We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

Destabilization of secondary structure in 16S ribosomal RNA by dimethylation of two adjacent adenosines.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.

Inhibition of elongation factor 2-dependent translocation by the pokeweed antiviral protein and ricin.

The pokeweed antiviral protein and the toxin ricin are two plant proteins which enzymatically inactivate eukaryotic ribosomes and cause the inhibition of the elongation cycle of protein synthesis. Their mode of inhibition of protein synthesis has been attributed to either the inhibition of enzymatic binding of aminoacyl-tRNA to the acceptor site of the ribosome or its subsequent translocation to the donor site. In this communication, we show that the treatment of ribosomes with either the pokeweed antiviral protein or ricin results in the inhibition of the elongation factor 2-catalyzed translocation of acceptor site aminoacyl-tRNA to the donor site. The inhibitory effects of both proteins are identical and are partially reversed by excess concentrations of elongation factor 2.

Characterization of the immunoprotective antigen of ribosomal preparations from Haemophilus influenzae

This investigation was designed to characterize the immunoprotective antigen of ribosomal preparations from Haemophilus influenzae. The ribosomes that elicited 80 to 90% protection contained 25% protein and 75% ribonucleic acid but did not contain any detectable hexoses. The immunodiffusion and hemagglutination inhibition tests also failed to demonstrate that the capsular material (polyribose phosphate) was in ribosomal preparations. Treatment of ribosomes with ribonuclease degraded 78% ribonucleic acid but did not affect the immunogenicity of such preparations. The proteolytic enzymes reduced the immunogenicity of ribosomes corresponding to the amount of protein degraded. The protection elicited by ribosomal protein extracted with 2-chloroethanol was comparable to that induced by intact ribosomes. In contrast, the low levels of protection observed by immunization with phenol-extracted ribonucleic acid were dependent on the amounts of contaminating protein. Finally, immunogenicity of ribosomal ribonucleic acid and protein was abrogated by treatment with proteolytic enzymes. These results clearly indicate that the protein associated with Haemophilus ribosomes is the major immunoprotective antigen.

The Plasmodium lophurae (avian malaria) ribosome.

Ribosomes were isolated from Plasmodium lophurae by Triton X-100 lysis and ultracentrifugation. Plasmodium lophurae ribosomal subparticles, produced by treatment of ribosomes with puromycin, were separated by sucrose gradient centrifugation. The nucleotide base composition of P. lophurae ribosomal RNA (rRNA) from intact ribosomes and subparticels was low in guanine-cytosine content (approximately 37% G + C) whereas the rRNA of the host cell (duck reticulocyte) was considerably higher (64% G + C). The rRNA of the malaria parasite was typically protozoan, with sedimentation values of 25S and 17S. In vitro, free parasites and malaria-infected red cells incorporated radioactive adenosine into both the large and the small plasmodial subparticles; label was also recovered in the 25S and 17S RNAs. These results suggest that host ribosomes do not contribute to ribosome biogenesis in this malaria, rather parasite rRNA is the transcription product of plasmodial cistrons.

Poly(4‐thiouridylic Acid) as Messenger RNA and Its Application for Photoaffinity Labelling of the Ribosomal mRNA Binding Site

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.

Comparison of the effects of bacteriophage T4 infection and N-ethylmaleimide on the translational specificity of Escherichia coli ribosomes

Treatment of ribosomes with N-ethylmaleimide (NEM) specifically inhibited the translation of f2 RNA by reacting with components of the high-salt wash fraction as well as the high-salt washed ribosomes themselves. The NEM-treated ribosomes were unable to efficiently bind fMet-tRNA at low concentrations of f2 RNA, but at higher concentrations the binding of initiator tRNA approached that of control ribosomes. The fMet-tRNA bound under the latter conditions could be completely released by puromycin. In spite of this, the NEM ribosomes could not efficiently translate even high concentrations of f2 RNA. Because the effects of NEM treatment mimic almost exactly those of bacteriophage T4 infection, in vitro translational specificity associated with phage infection may result from the oxidation of protein sulfhydryl groups either during infection or during subsequent isolation of the ribosomes.

Immunogenicity of Ribosomal Preparations from Yeast Cells of Histoplasma capsulatum

Protective immunity was elicited by immunization of mice with ribosomal preparations from yeast cells of Histoplasma capsulatum. Ribosomes from disrupted cells were isolated by differential centrifugation using sodium dodecyl sulfate. These preparations contained 55% protein and 45% ribonucleic acid and sedimented as a single peak with a sedimentation coefficient of 77S on sucrose density gradient analysis. Mice immunized subcutaneously with ribosomes, with or without adjuvant, were challenged intravenously with 8 x 10(6) yeast cells of H. capsulatum. Significant protection was induced by ribosomes and was greatly enhanced by adjuvants. Protection measured by 30-day survival compared favorably with the immunoprotection assessed by absence of lung lesions and negative spleen cultures. Treatment of ribosomes with ribonuclease before immunization reduced protection by 85%, whereas trypsin and Pronase reduced the protection by 50 to 55%. These findings indicate that both intact ribosomal ribonucleic acid and protein are necessary for maximal immunogenicity of Histoplasma ribosomes.

Conformational effects of mercurials on rat liver ribosomes: A comparison between the unmasking of a shielded protein in the 60S subunit by phenyl mercurials and edta

1. ( 1) Treatment of rat liver ribosomes with the mercurials p-hydroxymercuribenzoate (PHMB) or p-chloromercuriphenyl sulfonate (PCMPS), gives rise to characteristic alterations in the electrophoretic pattern of the proteins, indicating a binding of the reagents in situ to certain protein components. The reaction is reversed by mercaptoethanol or dithiothreitol (DTT). 2. ( 2) In the presence of 5–15 m M MgCl 2 (or lower concentrations of Ca 2+ or Mn 2+) the reaction with PHMB and PCMPS gives rise to a conformational alteration, by which a normally shielded protein in the large subunit (‘protein 5’) is made susceptible to chymotrypsin. The unmasking is reversed by mercaptoethanol or DTT, but cannot be effectively prevented by pretreatment of the ribosomes with SH-blocking reagents such as iodoacetate or N-ethylmaleimide (NEM). 3. ( 3) The activity of the ribosomes with respect to endogenous or poly U-directed phenylalanine incorporation is strongly inhibited by treatment with PHMB or PCMPS under the conditions of unmasking, but can be restored to a great extent with mercaptoethanol or DTT. During the period of mercurial-dependent unmasking the ribosomes are abnormally sensitive to inactivation by chymotrypsin, suggesting a functional importance of the unmasked chain(s). 4. ( 4) Reversible dissociation by current methods does not lead to the unmasking of protein 5. (Reversible dissociation by increased ionic strength leads to the unmasking of another protein in the 60S subunit, ‘protein 10’.) In contrast to the Mg 2+-stimulated unmasking by PHMB or PCMPS, the unmasking by Mg 2+ deprivation (ethylenediaminetetraacetate, EDTA) is irreversible. Although treatment of the ribosomes with PHMB or PCMPS under the conditions of unmasking of protein 5 may involve some dissociation of ribosomes to subunits, the unmasking is not dependent on this reaction. 5. ( 5) Neither the Mg 2+ stimulated unmasking of proteins by PHMB or PCMPS, nor the unmasking by Mg 2+-deprivation (EDTA) depends on RNA degradation or detachment of 5S RNA. The striking (although not complete) similarity between the two patterns of unmasking lends further support to the notion that the large subunit in mammalian ribosomes is predisposed to certain types of basic conformational reactions, which can be initiated by alternative and seemingly unrelated mechanisms.

Nucleases of membrane-bound rat liver ribosomes

Highly purified cytoplasmic membrane-bound ribosomes from rat liver, containing 63 % RNA and characterized by latent ribo- and deoxyribonuclease activity were isolated. On treatment of ribosomes with 500 mM LiCl, NaCl, and NH 4Cl solutions, which caused 15–20 % loss of the ribosomal protein, only a partial loss of the nuclease activity was observed. Crude, unpurified ribosomes revealed both 3′-ribonuclease and 5′-ribonuclease activities, their pH optima being 8.1 and 7.2, respectively. Apart from that, they possessed two kinds of deoxyribonuclease activity with pH optima of 8.1 and 5.3. As the purification process proceeded ribosomes lost their 5′-ribonuclease and acid deoxyribonuclease activities whereas the 3′-ribonuclease and alkali deoxyribonuclease specific activity slightly increased. Several lines of evidence clearly indicate that both ribonuclease and deoxyribonuclease activities are confined solely to small ribosomal subunits, the large ones being completely devoid of it. Latency can be ascribed only to the ribonuclease of intact ribosomes since the large subunit seems to protect the 18-S RNA molecule to a certain extent from attack by the enzyme residing in the small subunit as long as the structure of the whole ribosomal particle is preserved. On the other hand, nearly immediate degradation of the 18-S RNA molecule began in small separated subunits (in the medium optimal for the action of ribosomal ribonuclease) even while kept at 0°C. The experimental data presented in this paper do not support the alternative view that latency of ribosomal ribonuclease may be the result of an inactive dissociable complex formed by the enzyme and a ribosomal protein(s) with inhibitory properties. Enzyme preparations obtained by fractionation with (NH 4) 2SO 4 (0.4–0.8 satn) from purified ribosomes displayed both ribonuclease and deoxyribonuclease activity. Successive chromatography of these preparations on a DEAE-Sephadex A-50 column enabled us to separate the two activities. Both ribosomal enzymes seemed to be endonucleases, for even exhaustive enzyme hydrolysis of RNA and DNA yielded only relatively small mononucleotide fractions (10 and 4–5 %, respectively). The ribosomal ribonuclease appears to differ from pancreatic and alkali ribonuclease (a) in pH optimum, (b) in its action both on pyrimidine 2′:3′-cyclic phosphate and on poly(A) and poly(U) and (c) in the base composition of the “core” and mononucleotide fractions of the enzyme RNA hydrolyzate. Analysis of these fractions indicated that the phosphodiester bonds formed by adenine (AX) were the most resistant to the action of the ribosomal ribonuclease. On the other hand, the bonds most accessible to the enzyme proved to be pyrimidylpyrimidine and guanidylpyrimidine. As to the specificity of ribosomal deoxyribonuclease, the most readily hydrolyzed internucleotide bonds in the DNA molecule appear to be those formed by the thymidyl residues (XT). The enzyme displayed a slight preference for native DNA as the substrate.