Influence of magnesium and polyamines on the reactivity of individual ribosomal subunit proteins to lactoperoxidase-catalyzed iodination.

30S and 50S subunits, in the presence of either 20 mM Mg2+ or 6 mM Mg2+ and 5mM spermidine plus 25 mM putrescine, were observed to completely associate to form 70S monosomes as monitored by sucrose gradient sedimentation. Subunits maintained under the above ionic conditions were compared with 30S and 50S particles at low (6 mM) magnesium concentration with respect to the reactivity of individual ribosomal proteins to lactoperoxidase-catalyzed iodination. Altered reactivity to enzymatic iodination of ribosomal proteins S4, S9, S10, S14, S17, S19, and S20 in the small subunit of ribosomal proteins, L2, L9, L11, L27, and L30 in the large subunit following incubation with high magnesium or magnesium and polyamines suggests that a conformation change in both subunits accompanies the formation of 70S monosomes. The results further demonstrate that the effect of Mg2+ on subunit conformation is mimicked when polyamines are substituted for magnesium necessary for subunit association.     

The release and reassociation of 5.8 S rRNA with yeast ribosomes.

Yeast 5.8 S rRNA is released from purified 26 S rRNA when it is dissolved in water or low salt buffer (50 mM KCl, 10mM Tris-HCl, pH 7.5); it is not released from 60 S ribosomal subunits under similar conditions. The 5.8 S RNA component together with 5 S rRNA can be released from subunits or whole ribosomes by brief heat treatment or in 50% formamide; the Tm for the heat dissociation of 5.8 S RNA is 47 degrees C. This Tm is only slightly lower when 5 S rRNA is released first with EDTA treatment prior to heat treatment. No ribosomal proteins are released by the brief heat treatment. A significant portion of the 5.8 S RNA reassociates with the 60 S subunit when suspended in a higher salt buffer (e.g.0.4 m KCl, 25 mM Tris-HCl, pH 7.5, 6 mM magnesium acetate, 5 mM beta-mercaptoethanol). The Tm of this reassociated complex is also 47 degrees C. The results indicate that in yeast ribosomes the 5.8 S-26 S rRNA interaction is stabilized by ribosomal proteins but that the association is sufficiently loose to permit a reversible dissociation of the 5.8 S rRNA molecule.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

5 S RNA-protein complex is involved in ribosomal subunit association

The immobilized tRNA-50 S ribosomal subunit protein (TP50) complex binds the smaller ribosomal subunit. We constructed tRNA . TP50 . 5 S [32P] RNA and tRNA . TP50 . t [32P] RNA complexes and investigated the accessibility of the 32P-labelled tRNAs to ribonuclease T1. It was found that in this complex both 5 S RNA and tRNA are attacked by T1 RNase. In sharp contrast, the addition of 30 S subunit protects 5 S RNA as well as tRNA from degradation. We suggest that 5 S RNA-TP50 complex is exposed to the ribosomal interface and is involved in subunit interaction.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Sulfhydryl groups on yeast ribosomal proteins L7 and L26 are significantly more reactive in the 80 S particles than in the 60 S subunits.

The sulfhydryl-directed fluorescent reagent, 5-iodoacetamidofluorescein (IAF), reacts differently with proteins from the 60 S ribosomal subunit of Saccharomyces cerevisiae when this subunit is free as opposed to being contained within the 80 S ribosome. When the 80 S ribosomes and the free 60 S subunits were labeled with IAF, the specific fluorescence intensity (fluorescence intensity unit/A260 60 S subunit) of the subsequently derived 60 S was 16.3 and 5.4, respectively. Gel analysis showed that proteins L7 and L26 were selectively labeled and contained greater than 90% of the total fluorescent label, when 80 S ribosomes were labeled. When free 60 S subunits were labeled, six additional proteins were labeled. Both types of modified 60 S subunits were equally capable to support protein synthesis in vitro. Reassociation of the IAF-labeled derived and free 60 S subunits with unmodified 40 S subunits resulted in a maximum of 5-7% decrease and a 3-fold increase, respectively, in the fluorescence intensity without a shift in the emission maxima. The data suggest that ribosomal proteins L7 and L26 contain SH groups that respond to ribosomal subunit association and become more reactive in the intact ribosome than in the subunit. The environments of some or all of the additionally labeled proteins are also sensitive to subunit reassociation.     

Cooperative interactions among protein and RNA components of the 50S ribosomal subunit of Escherichia coli.

Copperative interactions among constituents of the 50S ribosomal subunit of Escherichia coli have been analyzed in order to elucidate its assembly and structural organization. Proteins L5 and L18 were shown to be necessary and sufficient to effect the association of the 5S and 23S RNAs into a quaternary complex that contains equimolar amounts of all four components. Measurement of diffusion constants by laser light scattering revealed that integration of the 5S RNA induced the 23S RNA to adopt a somewhat more open conformation. An investigation of relationships among proteins associated with the central and 3' portions of the 23S RNA demonstrated that attachment of L5, L10 + L11, and L28 depends upon the RNA-binding proteins L16, L2, and L1 + L3 + L6, respectively, and that L2 interacts with the central segment of the 23S RNA. These data, as well as the results of others, have been used to construct a scheme that depicts both direct and indirect associations of the 5S RNA, the 23S RNA, and over two-thirds of the subunit proteins. The 5' third of the 23S RNA apparently organizes the proteins required to nucleate essential reactions, whereas a region within 500 to 1500 bases of its 3' terminus is associated primarily with proteins involved in the major functional activities of the 50S ribosomal particle.     

The composition and properties of the Escherichia coli 5-S RNA-protein complex

At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg Cl2, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possess properties relevant to the function of the 50-S subunit; it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x 10(-7) M. The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits. The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichia coli ribosome.     

Covalent cross-linking of ribosomal RNA and proteins by methylene blue-sensitized photooxidation.

Ribosomal proteins are covalently cross-linked to ribosomal RNA by irradiation with visible light in the presence of methylene blue and O2. Proteins S3, S4, S5 and S7 from the 30 S subunit of Escherichia coli ribosomes and L2 and L3 from the 50 S subunit are among the cross-linked proteins. S3 and S5 had not previously been identified as RNA-binding proteins.     

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.     

Binding of Escherichia coli ribosomal proteins to 23S RNA under reconstitution conditions for the 50S subunit.

The RNA binding capacity of 50S proteins from E. coli ribosomes has been tested under improved conditions; purified proteins active in reconstitution assays were used, and the binding was studied under the conditions of the total reconstitution procedure for the 50S subunit. The results are: 1) Interaction of 23S RNA was found with 17 proteins, namely L1, L2, L3, L4, L7/L12, L9, L10, L11, L15, L16, L17, L18, L20, L22, L23, L24 and L29. 2) The proteins L1, L2, L3, L4, L9, L23 and L24 bound to 23S RNA at a level of about one copy per RNA molecule, whereas L20 could bind more than one copy (no saturation was observed at 1.8 copies per 23S RNA), and the other proteins bound 0.2--0.6 copies per RNA. 3) L1, L3, L7/L12 showed a slight binding to 16S RNA, L26 (identical with S20) strong binding to 16S RNA. 4) The binding of L2, L7/L12, L10, L11, L15, L16 and L18 was preparation sensitive, i.e. the binding ability changed notably from preparation to preparation. 5) All proteins bound equally well to 23S RNA in presence of 4 and 20 mM Mg2+, respectively, except L2, L3, L4, L7/L12, L9, L10, L15, L16 and L18, which bound less strongly at 20 mM than at 4 mM Mg2+.     

Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.     

Minimal set of ribosomal components for reconstitution of the peptidyltransferase activity.

A new approach is described to gain further information concerning the ribosomal components involved in the peptidyltransferase (PTF) activity exerted by Escherichia coli 50S subunits. A particle is reconstituted from highly purified proteins and RNA under modified incubation conditions. This particle contains only 16 out of the 34 distinct components constituting the native subunit, and yet still exhibits significant PTF activity. Single omission tests at the level of this "minimal ribosomal particle" indicate the limits set on a further reduction of the components, and in particular reveal that protein L18 can be excluded from the set of proteins which are essential for PTF activity, thus leaving L2, L3, L4, L15, and L16 as primary candidates for this function. 5S RNA is not needed for PTF activity of the "minimal ribosomal particle". Furthermore, a buffer condition is described which drastically improves the stability of total protein preparations and facilitates the isolation of individual proteins.     

Modifications of ribosomes from rat liver with alkylating derivatives of tRNA]

Alkylating analogs of peptidyl-tRNA: N-chloroambucilyl-14C-phenylanalyl-tRNA (1), N-iodoacetyl-14C-phenylalanyl-tRNA (2) and N-bromo-acetyl-14C-phenlalanyl-tRNA (3) were applied for the modification of the peptidyl-transferase center of the 80S ribosomes from rat liver. These analogs, being in the teronary complex poly-U: ribosome : tRNA analog, modified ribosomal proteins and ribosomal RNA. The modification is directed to large ribosomal subunit. It is found, that (1) modifies ribosomal proteins L5, L25, L31 and L32 and (2) modifies ribosomal proteins L4, L6, L10+L11, L13 and L30.     

cDNA nucleotide sequence and expression of a tobacco cytoplasmic ribosomal protein L2 gene.

The ribosomal protein L2 is an essential component of the ribosomal large subunit by its relation to the peptidyl transferase reaction, subunit association and elongation factor G-GTP binding. We have isolated a 937 nucleotide long cDNA encoding a cytoplasmic ribosomal L2 protein. Its deduced protein contains 260 amino acid residues and shows 65% identity with eucaryotic RL2 but only 32% identity with the chloroplast homologue. In addition, the protein presents the PROSITE signature which matches all the 50S and 60S L2 proteins and the two residues involved in the peptidyl transferase activity. The corresponding mRNA is accumulated in young plant tissues, in growing cell suspension and in germinating seeds but is not detectable in mature plant tissues, stationary cell suspension and in dry seeds. The mRNA accumulation is correlated with the growth process. Southern blot hybridization shows that cytoplasmic ribosomal protein L2 is encoded by two types of gene which could originate from each parent. highly homologous L2 genes were also detected by Southern blots in the genomes of several monocot and dicot plant species.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.