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.     

Characterization and properties of a 5S-RNA-protein complex released from heated 60S ribosomal subunits

When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.     

The ribosomal binding site for eukaryotic elongation factor EF-2 contains 5 S ribosomal RNA

The possible location of RNA in the ribosomal attachment site for the eukaryotic elongation factor EF-2 was analysed. Stable EF-2 · ribosome complexes formed in the presence of the non-hydrolysable GTP analogue GuoPP[CH₂]P were cross-linked with the short (4 Å between the reactive groups) bifunctional reagent, diepoxybutane. Non-cross-linked EF-2 was removed and the covalent factor-ribosome complex isolated. No interaction between EF-2 and 18 S or 28 S rRNA could be demonstrated. However, density gradient centrifugation of the cross-linked ribosomal complexes showed an increased density (1.25 g/cm³) of the factor, as expected from a covalent complex between EF-2 and a low-molecular-weight RNA species. Treatment of the covalent ribosome-factor complexes with EDTA released approx 50% of the cross-linked EF-2 from the ribosome together with the 5 S rRNA · protein L5 complex. Furthermore, the complex co-migrated with the 5S rRNA · L5 particle in sucrose gradients. Polyacrylamide gel electrophoresis showed that EF-2 was directly linked to 5 S rRNA in the 5 S rRNA · L5 complex, as well as in the complexes isolated by density gradient centrifugation. No traces of 5.8 S rRNA or tRNA could be demonstrated. The data indicate that the ribosomal binding domain for EF-2 contains the 5 S rRNA · protein L5 particle and that EF-2 is located in close proximity to 5 S rRNA within the EF-2 · GuoPP[CH₂]P · ribosome complex.     

Identification of homologous ribosomal proteins in HeLa cells and in Tetrahymena pyriformis. A study of proteins binding 5-S RNA and acidic proteins released from 40-S subunits by EDTA

Tetrahymena pyriformis 60-S ribosomal subunits treated with EDTA release a 7-S particle containing 5-S RNA and a 36000-Mr protein that is similar to mammalian 5-S-RNA-binding protein L5 in molecular weight, in two-dimensional acrylamide gel mobility, and in peptide pattern as generated by a simple, one-dimensional acrylamide gel technique. Human and T. pyriformis 40-S ribosomal subunits, treated with buffers lacking magnesium or containing EDTA, release varying amounts of two large acidic proteins. We have identified these released proteins by two-dimensional gel electrophoresis.     

5S RNA-protein complexes released by EDTA treatment of 60S ribosomal subunits of Tetrahymena thermophila

Treatment of large (60S) subunit of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila with EDTA causes quantitative release of 5S rRNA associated with variable non quantitative amounts of one or more of 60S proteins L4, L15, L24, L31 and L41. The composition of the group of proteins released with 5S rRNA depends on both the molar ratio of EDTA and 60S subunits and the concentration of 60S subunits, in treatment mixtures.     

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.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

Isolation and characterization of a 5S RNA-protein complex from human placental ribosomes

Large ribosomal subunits treated with EDTA change their sedimentation rate in sucrose gradients and lose the 5S RNA molecule, which is released in a ribonucleoprotein particle sedimenting at about 7S. The proteins bound in this complex were identified by two-dimensional (2-D) polyacrylamide gel electrophoresis as ribosomal proteins L3 and L4, both having a molecular weight of about 37000.     

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.     

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.     

Release of ribosome-bound 5S rRNA upon cleavage of the phosphodiester bond between nucleotides A54 and A55 in 5S rRNA

Reticulocyte lysates contain ribosome-bound and free populations of 5S RNA. The free population is sensitive to nuclease cleavage in the internal loop B, at the phosphodiester bond connecting nucleotides A54 and A55. Similar cleavage sites were detected in 5S rRNA in 60S subunits and 80S ribosomes. However, 5S rRNA in reticulocyte polysomes is insensitive to cleavage unless ribosomes are salt-washed. This suggests that a translational factor protects the backbone surrounding A54 from cleavage in polysomes. Upon nuclease treatment of mouse 60S subunits or reticulocyte lysates a small population of ribosomes released its 5S rRNA together with ribosomal protein L5. Furthermore, rRNA sequences from 5.8S, 28S and 18S rRNA were released. In 18S rRNA the sequences mainly originate from the 630 loop and stem (helix 18) in the 5' domain, whereas in 28S rRNA a majority of fragments is derived from helices 47 and 81 in domains III and V, respectively. We speculate that this type of rRNA-fragmentation may mimic a ribosome degradation pathway.     

Basepairing potential of the 3' terminus of 16S RNA: dependence on the functional state of the 30S subunit and the presence of protein S21.

The deoxyoctanucleotide 5'd (AAGGAGGT) which is complementary to the 3' terminus of 16S RNA has been used as a probe to measure the potential of this rRNA region to engage in intermolecular basepairing. The site specific binding of the octanucleotide is shown by labeling 16S RNA in situ at its 3' end with [32P]pCp and T4 RNA ligase (EC 6.5.1.3.). The label can be released as pA[32P]pCp by the simultaneous action of RNAse H (EC 3.1.4.34) and 5'd(AAGGAGGT). WE show that (1) 30S subunits prepared according to standard procedures, bind less than one copy of 5'd(AAGGAGGT); (2) isolated 16S RNA and 30S subunits inactivated by transcient exposure to 0.5 mM Mg2+ do not bind the octanucleotide; (3) binding to inactive subunits can be restored by a brief heat treatment; (4) 30S subunits lacking protein S21 do not bind 5'd(AAGGAGGT) even when submitted to heat treatment; (5) addition of protein S21 to subunits lacking S21 restores octamer binding; (6) the apparent exposure of the 16S RNA 3' terminus brought about by protein S21 is accompanied by the potential of the subunits to accept MS2 RNA as messenger; (7) the presence or absence of S1 on 30S subunits has no effect on their octanucleotide binding property.     

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.     

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5''-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.     

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.     

Modifications of 60 S ribosomal subunits induced by the ricin A chain

Incubation of 60 S ribosomal subunits with the ricin A chain reduced their stability during heat treatment. The toxin shifted the thermal denaturation curve of the subunits towards lower temperatures, in a similar way to that produced by the decrease in Mg²⁺ concentration. A brief heating (3 min at 57°C), which did not affect control subunit activity, enhanced protein synthesis inhibition of the toxin-treated subunits that released more 5 S RNA, in the form of nucleoprotein complex(es) with protein L5 and phosphoproteins P1P2 (RNP_H), than did heated control subunits [(1984) Eur. J. Biochem, 143, 303-307]. No nuclease activity tested on 60 S subunits and purified 5 S and 5.8 S RNA was found associated with the toxin. The results suggest that the toxin induced a limited conformational change of the 60 S subunit, which destabilized the interaction between RNP_H and the rest of the subunit.     

Studies of RNA-protein interactions in the yeast 5 S ribonucleoprotein particles by fluorescence and tritium exchange. Implications for ribosomal assembly

Studies of the conformational properties of the yeast 5 S RNA-protein complex were initiated in an attempt to understand loss of ability of its individual protein and RNA components to reassociate. The 5 S RNA-L1a protein complex from 60 S ribosomal subunits of Saccharomyces cerevisiae could be dissociated by high concentrations of magnesium. The degree of dissociation could be monitored by polyacrylamide gel electrophoresis. The complex was completely dissociated at about 390 mM magnesium, but was stable at 4 degrees C in 25 mM EDTA up to 48 h. The overall conformation of the complex was monitored using tritium exchange. The tritium exchange behavior was dramatically changed as the complex was dissociated. To determine contribution of each component to the observed overall change reflected in the tritium exchange behavior, ethidium bromide (EtBr) and bis-anilinonaphthalene-sulfonic acid fluorescence were used to monitor the RNA and the protein moiety, respectively. Upon dissociation of the complex, the fluorescence intensity resulting from EtBr binding to RNA decreased, whereas the intensity due to bis-anilinonaphthalene-sulfonic acid binding to the protein increased. Turbidity was observed during dissociation of the complex. These results indicate that disruption of interactions between the 5 S RNA and protein L1a resulted in an exposure of solvent-accessible apolar regions in the protein molecule. Such exposure led to insolubility of protein and irreversibility in interaction between individual components. Properties of the separated components also suggest that special conditions may be required for these components to associate during ribosomal assembly.     

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.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.