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.     

Mitochondrial and cytoplasmic ribosomes from mammalian tissues. Further characterization of ribosomal subunits and validity of buoyant-density methods for determination of the chemical composition and partial specific volume of ribonucleoprotein particles

1. At 0-4 degrees C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K(+) in the presence of 3.0mm-Mg(2+). When these subunits are placed in a medium containing a lower concentration of K(+) ions (25mm), approx. 75% of the subparticles recombine giving 55S monomers. 2. After negative staining the large subunits (20.3nm width) usually show a roundish profile, whereas the small subunits (12nm width) show an elongated, often bipartite, profile. The dimensions of the 55S ribosomes are 25.5nmx20.0nmx21.0nm, indicating a volume ratio of mitochondrial to cytosol ribosomes of 1:1.5. 3. The 39S and 29S subunits obtained in high-salt media at 0-4 degrees C have a buoyant density of 1.45g/cm(3); from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0x10(6) daltons and 1.20x10(6) daltons; the weights of the two subunits of cytosol ribosomes are 2.67x10(6) and 1.30x10(6) daltons. 4. The validity of the isodensity-equilibrium-centrifugation methods used to calculate the chemical composition of ribosomes was reinvestigated; it is confirmed that (a) reaction of ribosomal subunits with 6.0% (v/v) formaldehyde at 0 degrees C is sufficient to fix the particles, so that they remain essentially stable after exposure to dodecyl sulphate or centrifugation in CsCl, and (b) the partial specific volume of ribosomal subunits is a simple additive function of the partial specific volumes of RNA and protein. The RNA content is linearly related to buoyant density by the equation RNA (% by wt.)=349.5-(471.2x1/rho(CsCl)), where 1/rho(CsCl)=[unk](RNP) (partial specific volume of ribonucleoprotein). 5. The nucleotide compositions of the two subunit rRNA species of mitochondrial ribosomes from rodents (42% and 43% G+C) are distinctly different from those of cytoplasmic ribosomes.     

Specific interaction of proteins with 5 S RNA and tRNA in the 42 S storage particle of Xenopus oocytes

During early oogenesis in amphibia, most of the 5 S RNA and tRNA is stored in a ribonucleoprotein particle that sediments at 42 S. In Xenopus laevis the 42 S particle contains two major proteins: of Mr 48 000 (P48) and 43 000 (P43). It is shown that heterogeneity in composition of the 42 S particle reflects a changing situation whereby initially, both 5 S RNA and tRNA are complexed with P48 (1 molecule 5 S RNA: 1 molecule P48; 2 or 3 molecules tRNA: 1 molecule P48), but later, tRNA becomes increasingly associated with P43 (in a 1:1 ratio) although 5 S RNA remains complexed with a cleavage product of P48. These changes relate to the eventual utilization of the excess 5 S RNA and tRNA in ribosome assembly and protein synthesis.     

Specific interaction of proteins with 5 S RNA and tRNA in the 42 S storage particle of Xenopus oocytes

During early oogenesis in amphibia, most of the 5 S RNA and tRNA is stored in a ribonucleoprotein particle that sediments at 42 S. In Xenopus laevis the 42 S particle contains two major proteins: of M_r 48 000 (P48) and 43 000 (P43). It is shown that heterogeneity in composition of the 42 S particle reflects a changing situation whereby initially, both 5 S RNA and tRNA are complexed with P48 (1 molecule 5 S RNA: 1 molecule P48; 2 or 3 molecules tRNA: 1 molecule P48), but later, tRNA becomes increasingly associated with P43 (in a 1:1 ratio) although 5 S RNA remains complexed with a cleavage product of P48. These changes relate to the eventual utilization of the excess 5 S RNA and tRNA in ribosome assembly and protein synthesis.     

Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus.

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.     

RIBONUCLEOPROTEIN PARTICLES IN THE AMPHIBIAN OOCYTE NUCLEUS : Possible Intermediates in Ribosome Synthesis

Studies of the sedimentation properties of RNP¹ material from the nucleus of the amphibian oocyte have indicated (1) that there are few, if any, 78S ribosomes in the nucleus, (2) that there are smaller particles sedimenting at 50-55S and 30S, and (3) that the larger of these is the precursor of the 60S subunit of the cytoplasmic ribosomes. Although the nature of the 30S material is not completely clear, it probably includes precursor particles to the 40S ribosomal subunit. Heavy (50-55S) particles are predominant in immature oocytes of Triturus viridescens, whereas in immature oocytes of Triturus and Amblystoma mexicanum they are reduced greatly in amount, but are still detectable. Double-labeling studies of RNA and protein reveal that both types of particle incorporate uridine-³H, but that the 50-55S material of immature oocytes does not incorporate ¹⁴C-labeled amino acids. However, other evidence exists that favors the RNP nature of this material. Sedimentation analyses after SDS extraction show that 50-55S particles contain 40 and 30S RNA, whereas 30S particles contain 20S RNA. These types of RNA represent at least 80% of all the extractable nuclear RNA. The 50-55S particles are probably heterogeneous, including both particles containing mostly 40S RNA and particles containing only 30S RNA.     

Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes

The RNases P and MRP are involved in tRNA and rRNA processing, respectively. Both enzymes in eukaryotes are composed of an RNA molecule and 9–12 protein subunits. Most of the protein subunits are shared between RNases P and MRP. We have here performed a computational analysis of the protein subunits in a broad range of eukaryotic organisms using profile-based searches and phylogenetic methods. A number of novel homologues were identified, giving rise to a more complete inventory of RNase P/MRP proteins. We present evidence of a relationship between fungal Pop8 and the protein subunit families Rpp14/Pop5 as well as between fungal Pop6 and metazoan Rpp25. These relationships further emphasize a structural and functional similarity between the yeast and human P/MRP complexes. We have also identified novel P and MRP RNAs and analysis of all available sequences revealed a K-turn motif in a large number of these RNAs. We suggest that this motif is a binding site for the Pop3/Rpp38 proteins and we discuss other structural features of the RNA subunit and possible relationships to the protein subunit repertoire.     

Two zinc finger proteins from Xenopus laevis bind the same region of 5S RNA but with different nuclease protection patterns.

Immature oocytes from Xenopus laevis contain a 42S ribonucleoprotein particle (RNP) containing 5S RNA, tRNA, a 43 kDa protein, and a 48 kDa protein. A particle containing 5S RNA and the 43 kDa protein (p43-5S) liberated from the 42S particle upon brief treatment with urea can be purified by anion exchange chromatography. The purified p43-5S RNA migrates as a distinct species during electrophoresis on native polyacrylamide gels. Radiolabeled 5S RNA can be incorporated into the p43-5S complex by an RNA exchange reaction. The resulting complexes containing labeled 5S RNA have a mobility on polyacrylamide gels identical to that of purified p43-5S RNPs. RNP complexes containing 5S RNA labeled at either the 5' or 3' end were probed with a variety of nucleases in order to identify residues protected by p43. Nuclease protection assays performed with alpha-sarcin indicate that p43 binds primarily helices I, II, IV, and V of 5S RNA. This is the same general binding site observed for TFIIIA on 5S RNA. Direct comparison of the binding sites of p43 and TFIIIA with T1 and cobra venom nucleases reveals striking differences in the protection patterns of these two proteins.     

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.     

Roles of protein subunits in RNA-protein complexes: lessons from ribonuclease P

Ribonucleoproteins (RNP) are involved in many essential processes in life. However, the roles of RNA and protein subunits in an RNP complex are often hard to dissect. In many RNP complexes, including the ribosome and the Group II introns, one main function of the protein subunits is to facilitate RNA folding. However, in other systems, the protein subunits may perform additional functions, and can affect the biological activities of the RNP complexes. In this review, we use ribonuclease P (RNase P) as an example to illustrate how the protein subunit of this RNP affects different aspects of catalysis. RNase P plays an essential role in the processing of the precursor to transfer RNA (pre-tRNA) and is found in all three domains of life. While every cell has an RNase P (ribonuclease P) enzyme, only the bacterial and some of the archaeal RNase P RNAs (RNA component of RNase P) are active in vitro in the absence of the RNase P protein. RNase P is a remarkable enzyme in the fact that it has a conserved catalytic core composed of RNA around which a diverse array of protein(s) interact to create the RNase P holoenzyme. This combination of highly conserved RNA and altered protein components is a puzzle that allows the dissection of the functional roles of protein subunits in these RNP complexes.     

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.     

Sucrose modifies ribosomal stability and conformation

High concentrations of sucrose have a strong protective effect on heat-induced modifications of rat liver ribosomal subunits. They prevent to a large extent subunit inactivation, measured by poly (U)-dependent [14C] Phe tRNA binding (40S subunits) and puromycin reaction (60S subunits), subunit unfolding into light forms, and the release of both free and protein-complexed 5S RNA. They also increase the temperature at which subunits start to melt. Our data indicate that sucrose affects subunit conformation.