Studies on the conversion of 60s yeast ribosomes into a 50S component.
Ontology highlight
ABSTRACT:
PROVIDER: S-EPMC1185243 | BioStudies | 1969-01-01
REPOSITORIES: biostudies
Similar Datasets
Project description:The ribotoxin ?-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether ?-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, ?-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of ?-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a ?-sarcin cleaved SRL might be assessed only during translation.
| S-EPMC7293039 | BioStudies
Project description:Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.
1976-01-01 | S-EPMC1172712 | BioStudies
Project description:The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.
2006-01-01 | S-EPMC1500836 | BioStudies
Project description:1. The ;30s' and ;50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride-magnesium chloride solution, I 0.16, made 0-50mm with respect to Mg(2+). 2. Differentiation of enzymic and physical breakdown at Mg(2+) concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of ;50s' into 40-43s components. This was combined with the release of a small amount of ;5s' material which may be covalently bound soluble RNA. Other transformations of the ;50s' into 34-37s components were observed in both ribonuclease-active and -inactive ribosomes at 1.0-2.5mm-Mg(2+), and also with E. coli MRE600 when EDTA (0.2mm) was added to a solution in 0.16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg(2+) concentration 0.25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs(+)/Mg(2+) ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.
1965-01-01 | S-EPMC1207202 | BioStudies
Project description:DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.
| S-EPMC4900678 | BioStudies
Project description:Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.
1976-01-01 | S-EPMC1163988 | BioStudies
Project description:Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg(2+) exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.
| S-EPMC2770675 | BioStudies
Project description:Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these 'Mak5 cluster' factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.
| S-EPMC3846774 | BioStudies
Project description:Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.
| S-EPMC7411607 | BioStudies
Project description:1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.
1968-01-01 | S-EPMC1198493 | BioStudies