Sequential protein association with nascent 60S ribosomal particles

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.     

Subribosomal particles of cultured Chinese hamster cells : Identification of ring-shaped structures with 21S protein particles

The relationship of protein and ribonucleoprotein 18–21S subribosomal particles to 120 Å ring-shaped structures was determined. Such structures were found to co-sediment with the 21S protein particle and to concentrate with this particle during purification. Resolved 21S particles freed of 18S particles, LMM-RNPs, and 20S tRNP were shown to be comprised mainly of four or more proteins having molecular weights in the range of 22 000–28 000 D. Such proteins are completely absent from resolved 18S protein particles.     

General organization of protein in HeLa 40S nuclear ribonucleoprotein particles

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).     

Molecular mass, stoichiometry, and assembly of 20 S particles

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.     

Several crystal forms of the Bacillus stearothermophilus 50 S ribosomal particles

A kinetic analysis is presented of proton translocation, TMPD+ formation and oxidation of endogenous respiratory carriers during oxygen pulses of TMPD supplemented rat-liver mitochondria. The results show that antimycin-insensitive proton ejection observed under coupled conditions derives from oxidation of endogenous respiratory carriers and re-reduction of TMPD+ by hydrogenated donors and not from proton pumping by cytochrome oxidase as claimed by other investigators. The observations presented provide an example of certain interpretative difficulties in the use of redox mediators and of the methodological approaches that can be used to avoid these.     

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal particles.

Two-dimensional crystalline sheets of the large ribosomal subunit from Bacillus stearothermophilus have been obtained using a slightly modified procedure to that for growing three-dimensional crystals of the same material. The crystalline subunits are packed within monolayers in a relatively small unit cell, the dimensions of which are closely related to those observed for two forms of the three-dimensional crystals. The packing symmetry is p121, and the optical diffraction patterns of micrographs of negatively stained crystals extend to approximately 3.0 nm.     

Rotational diffusion of Escherichia coli ribosomes. I. - Free 70 S, 50 S and 30 S particles.

The rotational brownian diffusion of Escherichia coli ribosomes has been studied by following the transient dichroism generated by optical excitation of a covalent probe into its triplet state. The induced absorption anisotropy decays exponentially with characteristic correlation times: 2.5 microseconds, 1.6 microseconds and 1.1 microseconds for the 70S ribosome and the 50S and 30S subparticles respectively. The corresponding Stokes radii are in the same order, 133 A, 115 A and 103 A. The hydrodynamic properties are discussed in terms of an ellipsoidal shape of the ribosome particles.     

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.     

Polysomal and cytoplasmic mRNP particles containing 7S(L) RNA

Nuclear RNP complexes, cytoplasmic mRNP particles and free and membrane-bound polysomes were prepared from rat liver and their low-molecular-mass RNA components were analyzed on polyacrylamide/formamide gels. The separated small RNAs transferred to diazophenylthioether paper were hybridized to the nick-translated recombinant plasmid pA6 containing cDNA sequences for the low-Mr RNA called 7S(L) RNA. Nuclear RNP particles and free and membrane-bound polysomes were found to contain 7S(L) RNA. In the cytoplasm 7S(L) RNA could be identified as the major small RNA in 20-S cmRNP particles.     

40S hnRNP particles are a novel class of nuclear biomolecular condensates

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.     

Three-dimensional reconstruction of the 30 S ribosomal subunit from randomly oriented particles

Electron micrographs show the small (30 S) subunit of Escherichia coli ribosomes lying in a wide range of positions on the specimen support, related by rotation principally around the long axis of the particle. Through correspondence analysis, a multivariate statistical method that distinguishes the major factors accounting for interimage variance, the (aligned) views of the randomly oriented particles were ordered and grouped according to tilt angle. Views so grouped were then averaged and used as input to a three-dimensional reconstruction program.The particle reconstructed from nine averaged projections spanning a 160 ° rotational range has a resolution of 5 nm in planes perpendicular to the long axis of the particle and ~ 3 nm in the direction of the long axis. It is somewhat asymmetrical and quite compact; its most conspicuous feature is the “platform” that wraps partially around the middle of the subunit.     

Replication of double-stranded RNA in particles of Penicillium stoloniferum virus S.

RNA polymerase activity was assayed in different particle classes of Penicillium stoloniferum virus S. RNA polymerase activity was found to be associated with H particles, which contain double-stranded RNA and single-stranded RNA, but not with L particles, which contain only double-stranded RNA and not with M particles, which contain only single-stranded RNA. In H particles the reaction occurred with the formation of one new molecule of double-stranded RNA (or two complementary single strands of RNA) per virus particle and the production of product particles (P particles), which contained two molecules of double-stranded RNA (or its equivalent). This RNA polymerase is therefore a replicase, which catalyses the synthesis of the two complementary strands of double-stranded RNA in a single virus particle. This is the first report of this type of RNA polymerase system.     

The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy

The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis.     

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16

Ribosomal protein L16 was digested with Staphylococcus aureus protease V8 and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. One of the fragments, identified by sequence analysis as the N-terminal peptide of L16, was shown to exhibit partial peptide-bond-formation and transesterification activities of peptidyltransferase upon reconstitution with L16-depleted 50S core particles. However, several proteins enhanced these activities. L15 increased both reactions when added to the reconstitution mixture, suggesting a limited capacity of the L16 peptide to incorporate into 50S core particles. In contrast, the interaction of L11 with the N-terminal peptide stimulated the transesterification reaction but not the peptide-bond-forming activity of ribosomes, indicating a different topological domain for these reactions. Also, EF-P, a soluble protein which reconstructs the peptide-bond formation and transesterification reactions on 70S ribosomes, stimulated both peptidyltransferase activities exhibited by the L16 N-terminal peptide.