Association of nascent polypeptide with 30S ribosomal subunits

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg²⁺ concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).     

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.     

Association of nascent polypeptide and transfer ribonucleic acid with 30S ribosomal subunits

1. Crude extracts of Escherichia coli programmed in protein synthesis by endogenous mRNA have incorporated amino acids into protein. Analysis of such extracts by sucrose-gradient centrifugation in low Mg(2+) concentration has revealed that 30S ribosomal subunits carry associated radioactive material of which a considerable proportion can be removed from ribosomes by treatment of pre-labelled extracts with puromycin. 2. Gradient analyses of incorporations carried out in the additional presence of added (32)P-labelled tRNA have indicated that tRNA sediments in the regions of the newly synthesized nascent protein and that both labels are associated with all ribosomal components detected on the gradients under the experimental conditions employed. 3. 30S ribosomal subunits carrying both (32)P and (14)C labels have been isolated, disrupted with sodium dodecyl sulphate, and analysed by chromatography on Sephadex G-200 columns. Both labels elute closely together and well away from a tRNA marker analysed under identical conditions. 4. It is proposed that 30S ribosomal subunits, isolated from extracts which have synthesized nascent peptides under the direction of endogenous mRNA, carry associated peptidyl-tRNA.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Factors affecting nuclear export of the 60S ribosomal subunit in vivo

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the approximately 45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.     

Recessive nonsense-suppression in yeast: Involvement of 60S ribosomal subunit

Summary The ribosomal protein patterns of recessive suppressor strain and parent strain of Saccharomyces cerevisiae were analyzed by two-dimensional polyacrylamide gel electrophoresis. About 30 protein spots were found for ribosomal proteins of small subunit for both mutant and parent strain. These patterns do not differ from each other neither in intensity of staining, nor in mobility of spots. 41 protein spots were found in electrophoregrams of 60S ribosomal proteins both from parent strain and recessive suppressor strain. The electrophoretic picture of the 60S proteins from the parent and mutant strains is similar except the intensity of staining of the L30 spot. This protein is present in 60S subunit of suppressor strain and completely absent or only weakly stained on electrophoregrams of ribosomal proteins of parent strain. The possible relationships between the content of L30 protein and the mechanism of recessive suppression in yeast are discussed.     

Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit.

Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.     

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal subunits containing a nascent polypeptide chain

Polylysine chains were synthesized on Bacillus stearothermophilus ribosomes in a poly(A)-programmed in vitro system. After separation of the ribosomal subunits by sucrose gradient centrifugation, the polylysine chains (in contrast to the polyphenylalanine chains synthesized in a poly(U) system) reproducibly remained attached to the large ribosomal subunit. It was possible to produce two-dimensional crystalline sheets from the large ribosomal subunits containing the polylysine chains. These sheets are an essential prerequisite for three-dimensional reconstruction studies aiming to show that the tunnel in the large ribosomal subunit provides a path for the nascent polypeptide chain.     

The NOG1 GTP-binding protein is required for biogenesis of the 60 S ribosomal subunit

NOG1 is a nucleolar GTP-binding protein present in eukaryotes ranging from trypanosomes to humans. In this report we demonstrate that NOG1 is functionally linked to ribosome biogenesis. In sucrose density gradients Trypanosoma brucei NOG1 co-sediments with 60 S ribosomal subunits but not with monosomes. 60 S precursor RNAs are co-precipitated with NOG1. Together with the nucleolar localization of NOG1, these data indicate that NOG1 is associated with a precursor particle to the 60 S subunit. Disruption of NOG1 function through RNA interference led to a dramatic decrease in the levels of free 60 S particles and the appearance of an atypical rRNA intermediate in which ITS2 was not cleaved. Overexpression of mutant nog1 with a defect in its GTP binding motif on a wild type background caused a modest defect in 60 S biogenesis and a relative decrease in processing of the large subunit rRNAs. In contrast to the mutant protein, neither the N-terminal half of NOG1, which contains the GTP binding motifs, nor the C-terminal half of NOG1 associated with pre-ribosomal particles, although both localized to the nucleolus.     

Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit

Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.     

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.     

Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 Å away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S subunit before its first round of translation.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit

Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.     

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Assembly of the 30S ribosomal subunit

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.