Ribosomal Core Research Articles

Comparative ultrastructural ontogeny of zoosporangia of Zygorhizidium affluens and Z. planktonicum, chytrid parasites of the diatom Asterionella formosa

The fine-structural development of the small, 5–10 m, operculate zoosporangia of the monocentric chytrids Zygorhizidium affluens and Z. planktonicum , growing as epibionts on the planktonic diatom Asterionella formosa , is compared. In both species, following encystment and germination, the germ tube grows over the surface of the diatom frutule and penetrates the host cells by squeezing between the upper and lower girdle lamellae. As the cyst enlarges to form a sporangium, the single lipid globule breaks up and disperses, an operculum differentiates and the nucleus divides. In both species, cleavage of the cytoplasm into zoospore initials is mainly achieved by the infurrowing of the plasmamembrane, although there may be some contribution from internal cleavage vesicles. Flagellar axoneme profiles are not observed in the sporangia until cytoplasmic cleavage is well advanced. Specialized zoospore organelles, such as the rumposome fibrillar vesicles and endoplasmic reticulum-delimited ‘ribosome core’, do not form until the later stages of zoospore differentiation. The taxonomic and functional implications of these observations are discussed.

Structure function relationships in the ribosomal stalk proteins of archaebacteria.

The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.

Anti-P autoantibody production requires P1/P2 as immunogens but is not driven by exogenous self-antigen in MRL mice.

Considerable evidence supports the idea that autoantibody production in human and murine SLE is Ag driven. To determine whether Ag (the ribosomal P proteins) could initiate autoantibody production in lupus mice, 34 MRL/lpr mice were immunized with mouse riboosomal proteins in Freund's adjuvant. Neither intact ribosomes, denatured total mouse ribosomal proteins, nor the purified mouse ribosomal proteins, P1 and P2, induced the production of anti-P autoantibodies in the MRL/lpr mice. In contrast to these negative findings, MRL/lpr mice immunized with Artemia salina ribosomes produced anti-P antibodies as well as anti-P autoantibodies. Although the induced anti-P autoantibodies bound exclusively to the carboxyl terminus, these anti-P antibodies differed from spontaneously occurring anti-P autoantibodies in their predominant binding to mouse P0 on immunoblots and their preferential reactivity against A. salina synthetic peptides by ELISA. Induction of anti-P antibodies required the presence of P1 and P2 on the ribosome because ribosomal cores devoid of P1 and P2 dimers did not induce anti-P. Despite the presence of approximately 80 ribosomal proteins, autoantibodies to other mouse ribosomal proteins were rarely observed. Immunization of MRL/+ mice and a normal H-2-matched strain of mice, C3H, also resulted in anti-P antibodies reactive with the A. salina P proteins and mouse P0. Whereas anti-P levels gradually declined in C3H mice, anti-P levels either remained elevated (MRL/lpr) or showed a secondary rise (MRL/+) at the onset of autoimmunity. These observations indicate that: i) high levels of autologous Ag are not sufficient to drive antiribosomal autoantibody production in MRL mice, ii) multivalency of the P proteins may explain their potent immunogenicity and ability to break tolerance, and iii) immunized MRL mice show an abnormal persistence of high level anti-P production presumably reflecting T cell activation of presensitized B cells.

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.

Structure of methanogen genes

An analysis of the structure and presumed regulatory signals in sequenced methanogen genes is presented. The evidence for polycistronic transcriptional units is extended by inclusion of a DNA sequence, cloned from Methanobrevibacter smithii , which precedes the M. smithii pro C gene ( Hamilton and Reeve , 1985a). Comparison of DNA sequences indicates that a consensus core ribosome binding sequence for methanogens would be 5 'AGGTGA and that many of the ribosome binding sequences are, in fact, longer than the core sequence containing 7 bases with the potential to hybridize to 16SrRNA. The majority, but not all, of the sequenced methanogen genes conform to the rule in which RNY codons (R=purine, Y=pyrimidine, N=purine or pyrimidine) occur most frequently in the translated reading frame ( Shepherd 1981,1983). Codon usages by different methanogens reflect the need to accommodate genomes with very different overall %mol G+C contents. Codons such as AUA, AGA, and AGG, rarely used by E. coli are frequently used by methanogens. The dinucleotide, CG, which occurs very infrequently in eucaryotic DNAs ( Subak-Sharpe et al., 1967; Lennon and Faser , 1983; Nussinov , 1984) is also rarely found in methanogen genes. Methylation and demethylation of the cytosine residue in CG sequences has been implicated in regulation of eucaryotic gene expression ( Weisbrod , 1982) and this role used as an argument for the infrequent occurrence of CG-containing sequences. The rarity of CG in the DNA sequences so far obtained from methanogens may indicate a similar regulatory usage of CG-containing sequences in archaebacterial methanogens.

Localization of Lys-51 of L7/L12 on 50S ribosomes from Escherichia coli.

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys-51 with 4-(6-formyl-3-azido-phenoxy)butyrimidate. Reconstitution of ribosomal cores, lacking L7/L12, with imidate-modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation-factor-dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys-51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L12 stalk in such a way that Lys-51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.

The action of ricin A-chain on ribosomes

The mechanism of action of ricin, an irreversible inhibitor of protein synthesis in eukaryotic ribosomes, has been studied with ethanol extracted 80S rat liver ribosomes. The results show that the irreversible action of the toxin is on a component of the ribosome which remains in the core ribosome after the removal of the ethanol soluble proteins. The acid phosphoproteins P1 and P2 are shown not to be the site of action of the toxin.

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.

Factor requirements for the tritin inactivation of animal cell ribosomes

Wheat germ contains a protein (tritin) that efficiently inhibits protein synthesis in cell-free extracts from animal cells but not from wheat germ. Tritin has been purified to apparent homogeneity and shown to block enzymatically polypeptide chain elongation. We have extended these studies to examine more closely the mechanism of tritin inactivation of animal cell ribosomes. Here we provide evidence suggesting that ATP and tRNA, previously thought to be tritin co-factors, function in the inhibition by altering the conformation of the ribosome to a form susceptible to tritin attack. Tritin treatment does not inhibit the binding of aminoacyl-tRNA to ribosomes, but it does partially reduce the ribosome binding of elongation factor 2. Tritin inhibition appears to affect the core ribosome and not ribosome-associated factors. However, the core ribosome itself is not a suitable substrate for tritin attack; a factor(s) is removed from ribosomes by high salt washing which is required for the tritin-induced inhibition. The ability to be inhibited is restored when ascites cell core ribosomes are supplemented with factors from either ascites cells or wheat germ. In contrast, neither ascites cell factors nor wheat germ factors will promote a significant tritin-induced inhibition of core ribosomes from wheat germ. This indicates that the specificity of tritin inhibition resides primarily at the level of the eukaryotic core ribosome.

Hydrophobic interactions of Escherichia coli ribosomes

We have demonstrated the presence of hydrophobic sites on the surface of Escherichia coli ribosomes by means of hydrophobic chromatography on Octyl-Sepharose. Both 30-S and 50-S ribosomal subunits adsorb to Octyl-Sepharose at a low salt concentration, and can be eluted from it with a nonionic detergent without substantial changes in structure or activity. By testing a series of LiCl-derived ribosomal cores for their ability to adsorb to Octyl-Sepharose we have shown that the interaction of ribosomal particles with Octyl-Sepharose is dependent on the presence of certain ribosomal proteins; the core particles which lack these proteins do not bind to Octyl-Sepharose. The binding of a series of different ribosomal cores to nitrocellulose filters (Millipore) yielded the same pattern as was observed with Octyl-Sepharose, i.e. the more protein-depleted the particles, the less they were adsorbed. Thus, the adsorption of ribosomes to Millipore filters and to Octyl-Sepharose is presumably of the same hydrophobic nature.

Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6, N6-dimethyladenosine.

The modified nucleoside N6, N6-dimethyladenosine occurs in Escherichia coli 16S ribosomal RNA only in two successive positions near its 3' end. Antibodies directed against dimethyladenosine were induced with a nucleoside-albumin conjugate. As measured by second antibody precipitation of immune complexes, antidimethyladenosine antibodies bound 30S ribosomal subunits, ribosomal core particles, and ribosomal RNA which contain dimethyladenosine but showed little cross-reactivity with RNA or ribosomal subunits from a kasugamycin-resistant mutant which lacks dimethyladenosine. Antibody binding to ribosomal subunits was strongly influenced by the concentration of magnesium ion in the reaction medium and by the prior treatment of the subunits. Functionally active 30S subunits showed a striking binding optimum at 2-4 mM Mg2+; this optimum disappeared if the subunits were inactivated by dialysis against low concentrations of magnesium ion. Instead, the inactivated subunits showed a gradual increase in antibody binding as the magnesium ion concentration was raised to 20 mM; binding of 16S ribosomal RNA or subribosomal core particles from 30S subunits gave qualitatively similar curves, with no evidence of a low [Mg2+] optimum. The stability of antibody-subunit complexes was also found to depend upon subunit conformation and magnesium ion concentration; the half-life of an inactivated subunit-antibody complex (15 mM Mg2+) averaged 130 min, while active subunit-antibody complexes (3 mM Mg2+) had an average half-life of 70 min. More of the immune complexes with inactivated subunits were found to survive sucrose gradient sedimentation (relative to active subunits), and the concentration of subunits needed to halve antibody binding of [3H]-N6, N6-dimethyladenosine was lower with inactivated subunits. The results suggest that the antibody binding optimum seen with active subunits at 2-4 mM Mg2+ represents a dynamic aspect of the three-dimensional ribosomal subunit structure; a site near the 3' end of the RNA is involved, and both the availability of the modified nucleoside to an antibody probe and the stability of the resulting complexes are involved.

The role of ethanol extractable proteins from the 80S rat liver ribosome

80S rat liver ribosomes have been extracted with fifty percent ethanol at varying salt concentrations. The resulting 80S core ribosomes have lost almost all of their protein synthesis activity. The protein synthesis activity could be partially regained when the ethanol extracted proteins were reconstituted with the core ribosomes; however, reconstitution of the ribosome dependent EF-II GTP hydrolysis activity could not be detected. The ethanol extracts were found to contain only a few proteins, one or more of which we believe is necessary for the binding of elongation factor-II.

Studies on the RNA and protein binding sites of the E.coli ribosomal protein L10

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.

Inhibition of EF-G dependent GTPase by an aminoterminal fragment of L7L/12

The E. coli ribosomal proteins L12 and its N-acetylated form L7 were cleaved into an N-terminal and C-terminal fragment of roughly comparable size. The selective cleavage at the lone arginine residue was accomplished by trypsin treatment of the citraconylated proteins, followed by removal of the citraconyl moieties. These fragments, both separately and in combination, were incapable of reconstituting elongation factor G (EF-G) dependent GTPase of CsCl ribosomal cores supplemented with L10. However, incubation of cores containing L10 with the N-terminal fragment prevented the reconstitution of GTPase activity by intact L7/L12. No inhibition was observed when CsCl cores lacking L10 were incubated with the N-terminal fragment followed by addition of a preincubated mixture of L7/L12 and L10. The results indicate that the N-terminal part of L7/L12 is responsible for its ability to bind to 50S ribosomes and that L7/L12 together with L10 form a protein cluster on the ribosome.

Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes.

A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.

Assembly of ribosomal proteins L7, L10, L11, and L12, on the 50 S subunit of Escherichia coli.

We have determined the in vitro assembly sequence of ribosomal proteins L7, L10, L11, and L12 on Escherichia coli 50 S subunits by reconstitution experiments with the use of various ribosomal core particles and split protein fractions produced by treatment of 50 S subunits with 1 m NH4Cl and 50% ethanol. Proteins L7, L10, L11, and L12 were removed by a two-step treatment, first at 0 degrees, then at 37 degrees. Small amounts of proteins L1, L5, and L6 were also removed under these conditions. A one-step extraction of 50 S subunits at 0 degrees removed only proteins L7 and L12, while a similar one-step extraction of intact 50 S subunits at 37 degrees removed proteins L7, L12, and L10. Two-dimensional gel electrophoresis of the protein components and measurement of the ribosome-elongation factor G-guanosine diphosphate complex formed with the various reconstituted particles showed that the binding of proteins L7 and L12 is dependent on the binding of protein L10 and in turn, that the binding of protein L10 is dependent on the binding of protein L11.

The effect of β-ecdysone on protein synthesis in imaginal discs of Drosophila melanogaster cultured in vitro: II. Effects on synthesis in specific cell fractions

The effect of β-ecdysone on the synthesis of different proteins in imaginal discs of Drosophila melanogaster was investigated using double-label experiments and SDS acrylamide gel electrophoresis. Four different cellular fractions were analyzed (12K pellet, cytosol, core ribosomes, and KCl-soluble ribosomal proteins). β-Ecdysone alters the net synthesis of proteins in all of these fractions, with the most dramatic effects occurring in the ribosomal protein fractions. The maintenance of the altered pattern of synthesis of proteins in these fractions depends on the continued presence of β-ecdysone in the culture medium. The data suggest that β-ecdysone alters the synthesis of different proteins by acting on the genome. The apparent mechanisms by which β-ecdysone increases total protein synthesis and alters the net synthesis of different proteins in discs are discussed.

Activities of ribosomal cores deprived of proteins L7, L10, L11 and L12

Activities of ribosomal cores deprived of proteins L7, L10, L11 and L12

Alteration of ribosomal proteins and functions by 2-methoxy-5-nitrotropone.

The effect of 2-methoxy-5-nitrotropone on the ribosome has been studied. Treated particles are inactivated for polypeptide synthesis, aminoacyl-tRNA binding, and elongation-factor-dependent GTPase but catalize peptide bond formation. Treated particles (either 70-S ribosomes or ribosomal cores lacking proteins L7 and L12) recover their ability to hydrolyze GTP, but not to support polypeptide synthesis or aminoacyl-tRNA binding, upon the addition of these two untreated proteins. However, even in the absence of the added proteins the treated particles are able to carry out substantial GTP hydrolysis in the presence of 20% methanol. Interaction of particles with antibiotics such as chloramphenicol and thiostrepton is not affected by treatment. When proteins from treated 50-S subunits were analyzed by two-dimensional polyacrylamide gel electrophoresis, only proteins L3, and L15 were found not to be affected by the treatment. Proteins L1, L2, L4, L10, L13, L16, L17, L18, L22 and L24 showed spots of much less intensity than the controls and the other protein spots were not detected. A comparison of the resistant proteins with those present on CsCl-derived cores points to protein L15 as possibly involved in the peptidyl transferase centre of the ribosome.

Further studies with labelled polypeptide-chain-initiation factor 3 (IF-3).

The preparation of 35S‐labelled initiation factor 3 (IF‐3) from Escherichia coli is described. As reported earlier, labelled IF‐3 binds to 30‐S, but not to 50‐S ribosomal subunits or 70‐S ribosomes, and is released upon formation of a 70‐S couple. In continuation of this work we find that the 30‐S binding of IF‐3 is rather sensitive to aurin‐tricarboxylic acid, a powerful inhibitor of polypeptide chain initiation, but relatively insensitive to streptomycin. IF‐3 binds weakly to both 23‐S and 16‐S ribosomal RNA and to 23‐S but not 43‐S ribosomal cores, Free IF‐3 readily exchanges with the 30‐S‐bound factor even at low temperature. There is also an exchange of 50‐S or 30‐S ribosomal subunits labelled with [3H]uracil into 70‐S ribosomes in the absence of the reactions of protein synthesis. This exchange is markedly temperature‐dependent and strongly inhibited at high (20 mM) Mg2+ concentrations.