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.     

Effect of magnesium ion on the structure of the 5S RNA from Escherichia coli. An imino proton magnetic resonance study of the helix I, IV, and V regions of the molecule

The imino proton nuclear magnetic resonance spectrum of Escherichia coli 5S ribonucleic acid (RNA) changes when the Mg2+ ion concentration drops below physiological levels. The transition between the physiological and low magnesium spectral forms of 5S RNA has a midpoint at approximately 0.3 mM Mg2+. Many of the most conspicuous changes observed in the downfield spectrum of 5S RNA as the magnesium concentration is reduced are due to adjustments in the structures of helices I and IV and the disappearance of resonances originating in helix V. The binding of ribosomal protein L25 to 5S RNA in the absence of magnesium stabilizes helix V structures.     

Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments

The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.     

Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1-7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.     

Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes

Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized. One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272. Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits. However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes. Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes. Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation. Both antibodies inhibit the reassociation of 50 S with 30 S subunits. Both antibodies strongly inhibit peptidyltransferase activity. The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G). Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes. The Fab fragments of both antibodies had effects similar to the intact antibodies. The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.     

Three helical domains form a protein binding site in the 5S RNA-protein complex from eukaryotic ribosomes.

A ribosomal protein binding site in the eukaryotic 5S rRNA has been delineated by examining the effect of sequence variation and nucleotide modification on the RNA's ability to exchange into the EDTA-released, yeast ribosomal 5S RNA-protein complex. 5S RNAs of divergent sequence from a variety of eukaryotic origins could be readily exchanged into the yeast complex but RNA from bacterial origins was rejected. Nucleotide modifications in any of three analogous helical regions in eukaryotic 5S RNAs of differing origin reduced the ability of this RNA molecule to form homologous or heterologous RNA-protein complexes. Because sequence comparisons did not indicate common nucleotide sequences in the interacting helical regions, a model is suggested in which the eukaryotic 5S RNA binding protein does not simply recognize specific nucleotide sequences but interacts with three strategically oriented helical domains or functional groups within these domains. Two of the domains bear a limited sequence homology with each other and contain an unpaired nucleotide or "bulge" similar to that recently reported for one of the 5S RNA binding proteins in Escherichia coli (Peattie, D.A., Douthwaite, S., Garrett, R.A. and Noller, H.F. (1981) Proc. Natl. Acad. Sci. 78, 7331-7335). The results further indicate that the single ribosomal protein of eukaryotic 5S RNA-protein complexes interacts with the same region of the 5S rRNA molecule as do the multiple protein components in complexes of prokaryotic origin.     

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

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.     

Evolutionary changes in the higher order structure of the ribosomal 5S RNA.

Comparative studies have been undertaken on the higher order structure of ribosomal 5S RNAs from diverse origins. Competitive reassociation studies show that 5S RNA from either a eukaryote or archaebacterium will form a stable ribonucleoprotein complex with the yeast ribosomal 5S RNA binding protein (YL3); in contrast, eubacterial RNAs will not compete in a similar fashion. Partial S1 ribonuclease digestion and ethylnitrosourea reactivity were used to probe the structural differences suggested by the reconstitution experiments. The results indicate a more compact higher order structure in eukaryotic 5S RNAs as compared to eubacteria and suggest that the archaebacterial 5S RNA contains features which are common to either group. The potential significance of these results with respect to a generalized model for the tertiary structure of the ribosomal 5S RNA and to the heterogeneity in the protein components of 5S RNA-protein complexes are discussed.     

Nucleocytoplasmic transport of 5S ribosomal RNA

Nucleocytoplasmic transport of 5S ribosomal RNA in Xenopus oocytes occurs in the context of small, non-ribosomal RNPs. The complex with the zinc finger protein TFIIIA (7S RNP) is exported from the nucleus and stored in the cytoplasm, whereas the complex with the ribosomal protein L5 (5S RNP) shuttles between the nucleus and the cytoplasm. Nuclear import- and export-signals appear to reside within the protein moiety of these RNPs. Import of TFIIIA is inhibited by RNA binding, whereas nuclear transfer of L5 is not influenced by RNA binding. We propose that the export capacity of both, TFIIIA and L5, is regulated by the interaction with 5S ribosomal RNA.     

Ribosomal protein S1 induces a conformational change of the 30S ribosomal subunit

A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium bombardment method. Differences in exposure of some ribosomal proteins within the 30S subunit depleted of S1 and within the 30S–S1 complex were found. It was concluded that protein S1 binds in the region of the neck of the 30S ribosomal subunit inducing a conformational change of its structure.     

Initiation of mammalian protein synthesis: dynamic properties of the assembly process in vitro

Binding of the Met-tRNAMetf . eIf-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

Dissociation of intact Escherichia coli ribosomes in a mass spectrometer. Evidence for conformational change in a ribosome elongation factor G complex

We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg(2+) by Li(+) ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Analysis of a 5S RNA-protein complex isolated from the ribosomes of rye embryos

Rye embryo ribosomes were dissociated into subunits and the large subunit fraction was treated with formamide. A low molecular weight complex of RNA and protein (RNP) was released. Electrophoresis of the RNP in polyacrylamide gels containing sodium dodecyl sulphate yielded an RNA band and a single protein band. The protein had a molecular weight of approximately 41 000 and the RNA of the complex was shown to be 5S ribosomal RNA. Embryos were germinated in the presence of [32P]orthophosphate and the labelled RNP was isolated from their ribosomes. The RNA component was partially digested with pancreatic A ribonuclease and the parts protected from degradation by the protein were determined by sequence analysis. Although the whole 5S RNA molecule was shielded to some extent, the portion most protected was between nucleotides 68 and 108. This is, therefore, probably the part of plant cytosol 5S RNA which is primarily involved in the interaction with protein in the complex and possibly in the ribosome as well.     

Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA

A library of random mutations in Xenopus ribosomal protein L5 was generated by error-prone PCR and used to delineate the binding domain for 5S rRNA. All but one of the amino acid substitutions that affected binding affinity are clustered in the central region of the protein. Several of the mutations are conservative substitutions of non-polar amino acid residues that are unlikely to form energetically significant contacts to the RNA. Thermal denaturation, monitored by circular dichroism (CD), indicates that L5 is not fully structured and association with 5S rRNA increases the t(m) of the protein by 16 degrees C. L5 induces changes in the CD spectrum of 5S rRNA, establishing that the complex forms by a mutual induced fit mechanism. Deuterium exchange reveals that a considerable amount of L5 is unstructured in the absence of 5S rRNA. The fluorescence emission of W266 provides evidence for structural changes in the C-terminal region of L5 upon binding to 5S rRNA; whereas, protection experiments demonstrate that the N terminus remains highly sensitive to protease digestion in the complex. Analysis of the amino acid sequence of L5 by the program PONDR predicts that the N and C-terminal regions of L5 are intrinsically disordered, but that the central region, which contains three essential tyrosine residues and other residues important for binding to 5S rRNA, is likely to be structured. Initial interaction of the protein with 5S rRNA likely occurs through this region, followed by induced folding of the C-terminal region. The persistent disorder in the N-terminal domain is possibly exploited for interactions between the L5-5S rRNA complex and other proteins.     

The MDM2 oncoprotein binds specifically to RNA through its RING finger domain.

BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.     

Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.