Defects In Ribosome Synthesis Research Articles

Pre-Ribosomal RNA Processing in Human Cells: From Mechanisms to Congenital Diseases.

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

Fitness advantages conferred by the L20-interacting RNAcis-regulator of ribosomal protein synthesis inBacillus subtilis

In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5′-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis.These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA.

Mitochondrial Dysfunction As a Potential Source of Reactive Oxygen Species in Cellular Models of Shwachman-Diamond Syndrome

Abstract 1343Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology. Disclosures:No relevant conflicts of interest to declare.

PIM1 kinase is destabilized by ribosomal stress causing inhibition of cell cycle progression

PIM1 is a constitutively active serine/threonine kinase regulated by cytokines, growth factors and hormones. It has been implicated in the control of cell cycle progression and apoptosis and its overexpression has been associated with various kinds of lymphoid and hematopoietic malignancies. The activity of PIM1 is dependent on the phosphorylation of several targets involved in transcription, cell cycle and apoptosis. We have recently observed that PIM1 interacts with ribosomal protein (RP)S19 and cosediments with ribosomes. Defects in ribosome synthesis (ribosomal stress) have been shown to activate a p53-dependent growth arrest response. To investigate if PIM1 could have a role in the response to ribosomal stress, we induced ribosome synthesis alterations in TF-1 and K562 erythroid cell lines. We found that RP deficiency, induced by RNA interference or treatment with inhibitor of nucleolar functions, causes a drastic destabilization of PIM1. The lower level of PIM1 induces an increase in the cell cycle inhibitor p27(Kip1) and blocks cell proliferation even in the absence of p53. Notably, restoring PIM1 level by transfection causes a recovery of cell growth. Our data indicate that PIM1 may act as a sensor for ribosomal stress independently of or in concert with the known p53-dependent mechanisms.

Distinct ribosome maturation defects in yeast models of Diamond-Blackfan anemia and Shwachman-Diamond syndrome

Diamond-Blackfan anemia and Shwachman-Diamond syndrome are inherited bone marrow failure syndromes linked to defects in ribosome synthesis. The purpose of this study was to determine whether yeast models for Diamond-Blackfan anemia and Shwachman-Diamond syndrome differed in the mechanism by which ribosome synthesis was affected. Northern blotting, pulse-chase analysis, and polysome profiling were used to study ribosome synthesis in yeast models. Localization of 60S ribosomal subunits was assessed using RPL25eGFP. Relative to wild-type controls, each disease model showed defects in 60S subunit maturation, but with distinct underlying mechanisms. In the model of Diamond-Blackfan anemia, 60S subunit maturation was disrupted at a relatively early stage with abortive complexes subject to rapid degradation. 5S ribosomal RNA, unlike other large subunit ribosomal RNA in this model, accumulated as an extra-ribosomal species. In contrast, subunit maturation in the Shwachman-Diamond syndrome model was affected at a later step, giving rise to relatively stable pre-60S particles with associated 5S ribosomal RNA retained in the nucleus. Conclusions These differences between the yeast Diamond-Blackfan anemia and Shwachman-Diamond syndrome models have implications for signaling mechanisms linking abortive ribosome assembly to cell fate decisions and may contribute to the divergent clinical presentations of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

Anémie de Diamond-Blackfan

Diamond-Blackfan anemia (DBA), a rare congenital erythroblastopenia, has recently become a paradigm for a growing set of genetic diseases linked to mutations in genes encoding ribosomal proteins or factors involved in ribosome biogenesis. Recent studies of the structure and the function of ribosomal proteins affected in DBA indicate that their mutation in DBA primarily impacts ribosome biogenesis. Accordingly, cells from DBA patients display anomalies in the maturation of ribosomal RNAs. The explanation of this unexpected link between ribosome biogenesis, a ubiquitous process, and a disease mostly affecting erythroid differentiation may stem in part from the emerging concept of ribosomal stress response, a signaling pathway triggering cell cycle arrest in response to a defect in ribosome synthesis. Future studies of DBA and other diseases related to defects in ribosome biogenesis are likely to rapidly provide important insights into the regulatory mechanisms linking cell cycle progression to this major metabolic pathway.

Yeast Models of Diamond Blackfan Anemia and Shwachman Diamond Syndrome Differ in Their Effect on the Synthesis and Function of Ribosomal Subunits.

Nucleolar stress is a frequently invoked mechanism used to describe the pro-apoptotic phenotype of cells affected in human diseases linked to abnormalities of the ribosome. However, the diversity of clinical phenotypes observed in these diseases suggests that there may be different types of nucleolar and/or translational stress that stimulate cell death pathways by alternative mechanisms. We have studied yeast models of Diamond Blackfan anemia (DBA) and Shwachman Diamond syndrome (SDS), two inherited bone marrow failure syndromes linked to defects in ribosome synthesis and/or function, to determine potential underlying molecular mechanisms that distinguish these disease models. To date, all genes identified in DBA encode ribosomal proteins. In contrast, SBDS, the gene affected in SDS encodes a protein that associates with 60S subunits, but is not considered a structural component of the ribosome. We have analyzed the translational capacity of cells harboring mutations in RPL33A and SDO1, yeast orthologs of genes affected in DBA and SDS, respectively. Polysome profiles from cells depleted of Rpl33A have a decrease in the amount of free 60S subunits and the presence of half-mer polysomes, as expected for an essential structural component of the 60S subunit. Polysome profiles from cells depleted of Sdo1 also had half-mer polysomes, but in this case there were significant amounts of free 60S subunits evident. Analysis of the intracellular distribution of 60S subunits by fluorescence microscopy revealed significant differences between the two disease models. In the DBA model, there was no evidence of accumulation of incompletely assembled subunits in the nucleolus indicating that rapid degradation. In contrast, in the SDS model there was significant accumulation of 60S subunits in the nucleoplasm. Thus, the two disease models interfere with the biogenesis of 60S subunits through distinct mechanisms. To determine if these mechanistic differences influence protein synthesis, we analyzed the patterns of proteins synthesized in these two disease models. We found that the expression of the 20S replicon was induced in both models, a sign of general translational stress. However, the two models also showed distinct differences in the synthesis of certain proteins. Thus, the mechanisms by which reductions of Rpl33A or Sdo1 influence levels of functional 60S subunits have differential effects on the patterns of proteins synthesized within cells Together these data indicate that the ribosome-based diseases may result from a composite of effects that include both nucleolar stress mechanisms and changes in translational output. The distinct clinical phenotypes observed in these disorders may result from differences in the relative contributions of either of these two mechanisms.

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.

Surveillance of nuclear-restricted pre-ribosomes within a subnucleolar region of Saccharomyces cerevisiae.

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.

Translation Initiation Defect and Gene Profiles Linked to Depletion of Shwachman-Diamond Syndrome Gene Product.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to leukemia. Mutations in SBDS have been shown to cause SDS, and SBDS's yeast ortholog, SDO1, appears to play a role in ribosomal RNA (rRNA) processing. Thus, several bone marrow failure syndromes including dyskeratosis congenita, cartilage hair hypoplasia, and Diamond Blackfan anemia have been linked to defects in ribosome synthesis. In order to clarify the function of SBDS, we have performed studies on the effect of depleting SBDS in yeast, mouse, and human cells. Six plasmids encoding for coral GFP and neomycin resistance were modified to express siRNAs against SBDS and used to establish stable transfectants in human HeLa and mouse NIH3T3 cells. Each siRNA was confirmed to knock down human or mouse SBDS expression, by real-time PCR. The growth of HeLa and NIH3T3 stable transfectants was markedly hindered when compared to that of cells stably transfected with a siRNA against an irrelevant control gene. The growth abnormality in SBDS-knockdown cells appeared to be related to an increased propensity to apoptosis. We then searched for differentially expressed genes between SBDS-knockdown and control cells using a series of 9 Affymetrix GeneChip microarrays. An exhaustive comparison using GenMAPP 2.0 and Gene Ontology software yielded highly significant differences in expression of genes in multiple pathways such as: apoptosis, cell cycle and cell growth, neutrophil chemotaxis, skeletal development, and angiogenesis pathways, among others. Changes (resulting from SBDS silencing) in expression of ribosome-related genes were also evident across experiments with both human and mouse cells. Concurrently, we studied the effect of depleting the yeast ortholog of SBDS, known as SDO1. Mutants of SDO1 are deficient in early steps in rRNA processing resulting in the accumulation of 35S and 23S pre-rRNA. SDO1 mutants thus have a so-called processome-like phenotype, in which cleavages at sites A0 and A1 within the external transcribed sequence-1 and site A2 within the internal transcribed sequence-1 are severely affected. Processome mutants exhibit a selective deficit in 40S ribosomal subunits, as cleavages at sites A0, A1, and A2 are not absolutely required for the maturation of 60S ribosomal subunits. Surprisingly, we found a much more complex pattern than anticipated in polysome profiles from cells harboring null alleles of SDO1. These profiles contained half-mer polysomes, typical of strains with a 60S subunit deficit and yet, in SDO1 mutants the amount of 60S subunits appeared to be relatively normal. In addition, the pool of free 40S subunits appeared abnormal in SDO1 mutants, with the appearance of two peaks, one of which likely represents an aberrant 40S subunit. This aberrant 40S subunit was apparently capable of early steps in translational initiation but failed to join with 60S subunits to form the 80S initiation complex, resulting in half-mer polysomes. We conclude that the rRNA processing phenotypes of SDO1 mutants are likely secondary to a defect in recruiting a factor or factors to the 40S subunit necessary for subunit joining during initiation. A similar profile for free 40S subunits was seen in NIH3T3 cells in which Sbds had been knocked down, suggesting that at least a part of the molecular phenotype observed in yeast is directly relevant for mammalian cells.

Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins

The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5-10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

Ribosomal Protein S19 and Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is one of several bone marrow failures that have been linked to defects in ribosome synthesis. 25% of DBA cases are linked to mutations in ribosomal protein S19 (Rps19). The etiology of the remaining cases is unknown. To gain a better understanding of the function of the Rps19 family of proteins we have characterized members of this protein family in the yeast, Saccharomyces cerevisiae. In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B. Yeast cells lacking both RPS19 genes are not viable, whereas those lacking a single gene are viable but have growth defects. These latter strains are defective in a specific step in rRNA processing that preferentially affects the maturation of 40S ribosomal subunits. We scanned other yeast strains with mutations in genes for 40S subunit proteins for processing phenotypes similar to RPS19 mutants. Several have phenotypes that overlap with RPS19 mutants, but only RPS18 stands out as being virtually identical to RPS19 mutants. The human RPS18 gene is therefore a candidate locus for pathogenic mutations in DBA patients with normal RPS19. We are currently developing strategies to sequence RPS18 genes from DBA patients with normal RPS19 to determine if mutations in RPS18 are associated with DBA. We have also developed a yeast system for the functional testing of mutant alleles of RPS19 found in DBA patients. In general, a mutation is considered pathogenic if it is not found in unaffected family members and in the general population. We have found, however, that several missense mutations classified as pathogenic in DBA patients do not affect Rps19 function in the yeast system. The failure of these mutations to affect Rps19 function in yeast points to a need for functional testing of RPS19 mutant alleles in human cells.

Ribosomal RNA and Gene Expression Abnormalities with Knockdown of the Shwachman-Diamond Syndrome Gene, SBDS.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to myelodysplastic syndrome and acute myeloid leukemia. Recently, mutations in an uncharacterized gene, SBDS, have been associated with SDS. SBDS is a member of a highly conserved protein family of unknown function, with putative orthologs in species ranging from archaea to plants and vertebrates. The yeast ortholog, YLR022c, is a phospholipid-binding protein that is essential for cell viability and may play a critical role in ribosomal RNA (rRNA) processing. Several bone marrow failure syndromes including dyskeratosis congenita, cartilage hair hypoplasia, and Diamond Blackfan anemia (DBA) have been linked to factors involved in ribosome synthesis. Our studies in yeast show that rRNA processing phenotypes associated with mutations in RPS19, a gene affected in DBA, overlap with the rRNA processing phenotype reported by others for cells depleted of YLR022c. These data indicate that defects in ribosome synthesis may contribute to the molecular basis of SDS. To study the effect of depleting SBDS in human cells, we created plasmids encoding for coral GFP (cGFP) and neomycin resistance that were further modified to express siRNA against human SBDS. A construct that contains only cGFP/NeoR was used as a control. HeLa cells were transiently transfected with knockdown or control vector for two days and flow-sorted for cGFP expression. In some experiments, neomycin- resistant siRNA expressing cell clones were isolated. Total RNA was extracted from transfected cells, reverse-transcribed, and subjected to real-time PCR for SBDS transcript quantification (using two SBDS primers and a TaqMan 5′ Fam/3′ Tamra probe specific for SBDS transcript). Data from each transfection were normalized to GAPDH expression, and SBDS expression was compared between cells treated with each siRNA vector and cells treated with a control cGFP/NeoR construct lacking the siRNA sequence. After multiple preliminary experiments, we found that one of the siRNAs expressed in cGFP-positive sorted cells decreased SBDS expression to 16% that of cells expressing only cGFP but not the siRNA sequence (84% knock-down). Total RNA was extracted from cGFP-positive knockdown and control cells and analyzed by Affymetrix GeneChip U133A microarrays to search for differentially expressed genes. In general, comparing the knockdown and control cells, we found many more genes with increased rather than decreased expression. However, both 28S and 18S rRNAs were repeatedly found significantly decreased in the knockdown cells (5–10% that of cGFP-positive control cells lacking the siRNA sequence). Although human rRNA processing is not fully understood, inhibition of early processing steps in mammalian cells should affect the production of both 18S and 28S rRNAs. Thus, our results suggest that at least one function of SBDS may be in rRNA processing and furthers the putative link between certain congenital bone marrow failure syndromes and defective ribosomal biogenesis.

Identification of a role for Saccharomyces cerevisiae Cgr1p in pre-rRNA processing and 60S ribosome subunit synthesis.

Saccharomyces cerevisiae CGR1 encodes a conserved fungal protein that localizes to the nucleolus. To determine if this localization reflects a role for Cgr1p in ribosome biogenesis two yeast cgr1 mutants were examined for defects in ribosome synthesis: a conditional depletion strain in which CGR1 is under the control of a tetracycline-repressible promoter and a mutant strain in which a C-terminal truncated Cgr1p is expressed. Both strains had impaired growth rates and were hypersensitive to the aminoglycosides paromomycin and hygromycin. Polysome analyses of the mutants revealed increased levels of free 40S subunits relative to 60S subunits, a decrease in 80S monosomes and accumulation of half-mer polysomes. Pulse-chase labelling demonstrated that pre-rRNA processing was defective in the mutants, resulting in accumulation of the 35S, 27S and 7S pre-rRNAs and delayed production of the mature 25S and 5 small middle dot8S rRNAs. The synthesis of the 18S and 5S rRNAs was unaffected. Loss of Cgr1 function also caused a partial delocalization of the 5'-ITS1 RNA and the nucleolar protein Nop1p into the nucleoplasm, suggesting that Cgr1p contributes to compartmentalization of nucleolar constituents. Together these findings establish a role for Cgr1p in ribosome biogenesis.

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa.

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22 degrees C, the un-18 mutation did not affect the period length, but at temperatures between 22 degrees C and 32 degrees C, the period length of the un-18 mutant was approximately 2 h longer than that of the wild-type strain. The un-18+ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora.

Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.

An unusual precursor of 50S ribosomes in a mutant of Escherichia coli.

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.

A genetic analysis of a mutant of Escherichia coli with a defect in the assembly of ribosomes.

The complex phenotype of a mutant of Escherichia coli with a defect in the assembly of ribosomes has been shown to be due to the presence of two mutations, both of which are closely linked to the gene (xyl) that determines the ability to ferment xylose. One of the mutations is responsible for the defect in ribosome synthesis, whilst the other is associated with an altered degree of resistance to streptomycin and a reduced efficiency in the utilisation of the growth substrate.