Pool Of Ribosomes Research Articles

Depth profile of sulfate-reducing bacterial ribosomal RNA and mercury methylation in an estuarine sediment

The community structure of complex anaerobic microbial communities has been difficult to elucidate because of an inability to cultivate most of the contributing populations. In this study, the distribution of sulfate-reducing bacteria (SRB) in anaerobic sediments was determined using oligonucleotide probes complementary to the 16S ribosomal RNAs of major phylogenetic groups. Sediment cores were collected from Santa Rosa Sound in northwest Florida, and sectioned by depth into 1 to 2 cm fractions. Nucleic acids were extracted from each fraction and hybridized with the SRB-specific ribosomal RNA probes. SRB ribosomal RNAs accounted for almost 5% of the microbial community ribosomal RNA pool in the 3–4 cm depth fraction and were dominated by Desulfovibrionaceae ribosomal RNA. The SRB ribosomal RNA peak coincided with mercury methylation, an activity attributed to SRB. Profiles of the ribosomal RNAs indicate that SRB populations in sediments are stratified by depth.

Specialized Ribosomes Allow for the Study of Mutations in Functionally Important Regions in 16 S rRNA, without Affecting Cell Growth: The Identification of Functional Regions in the Central Domain of 16 S rRNA

In order to identify the sequences in the central domain of 16 S rRNA of Escherichia coli that are important for ribosome function, we have generated random mutations using a PCR-based mutagenesis technique. We show that the effects of such mutations on ribosomal activity can be analyzed in vivo utilizing the specialized ribosome system. With this system the effect of rRNA mutations on ribosomal activity can be studied by measuring the translation of a modified CAT-mRNA by specialized ribosomes. Specialized ribosomes do not translate the endogenous mRNAs and, therefore, are expected to constitute a nonessential pool of ribosomes within the cell. In total, we have isolated 28 different clones harboring specialized ribosomes with single or multiple point mutations. We demonstrate that for none of these clones was cell growth retarded, even though some of the mutations severely impaired the activity of the specialized ribosomes, to as low as 3% of the wild-type level. For all mutants, their individual activities ranged between 3% and 100% of that of the wild-type activity. Comparison of several mutants indicates that mutations within the hairpin loops 787-795 and 898-901 strongly reduce the ribosomal activity. We also present evidence that the single-stranded region centered around residue A815 may be involved in maintaining translational accuracy.

The Escherichia coli ribosomal RNA leader: a structural and functional investigation.

The structure of the E. coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification. The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model. The binding of purified Nus proteins to the ribosomal leader RNA was further tested. Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC. The possible significance of this interaction is discussed. In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo. A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant). It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared. High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions. Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation. The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.

Coupling of rRNA Transcription and Ribosomal Assembly in Vivo: Formation of Active Ribosomal Subunits in Escherichia coli Requires Transcription of rRNA Genes by Host RNA Polymerase which Cannot be Replaced by Bacteriophage T7 RNA Polymerase

Transcription of a plasmid-located rrnB operon and the corresponding formation of ribosomes in vivo were studied using either T7 RNA polymerase or host RNA polymerase as transcriptase. The 23 S rRNA gene on the plasmid carried an A1067 → T mutation, which confers resistance against the drug thiostrepton. The proportion of particles containing plasmid-borne 23 S rRNA versus chromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067. The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes. When the plasmid rrnB operon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded. The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e. particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes. The particles with plasmid-derived rRNAs were also practically inactive in protein synthesis in vitro . However, the rRNA was functional as shown by reconstitution analysis. The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction or rRNA but rather the T7 transcriptase was responsible for the observed effects. However, when the plasmid rrnB operon was transcribed with host RNA polymerse, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro. When the induction of rRNA transcription by T7 RNA polymerase was performed at 25°C instead of 37°C, an almost normal pattern was observed. Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes. Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37°C than the host polymerase. The results suggest that the formation of active ribosomal subunits in vivo requires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assembly in vivo. T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37°C.

In vivo effect of l-leucine administration on protein synthesis in mice

To clarify the in vivo effect of leucine on protein synthesis within a short period, the fractional formation rate of peptidyl puromycin (FFR) was measured in the liver and skeletal muscle of mice. FFR was calculated as the ratio of specific activity of peptide-bound puromycin at 10 minutes after injection of [ 3H]puromycin to the area under the specific activity-time curve of free puromycin. The value of FFR corresponds to the amount of tracer puromycin bound in unit time, expressed as a fraction of the total amount of puromycin potentially bound to the aminoacyl-site on the functional ribosome pool. In 12-hour fasted, streptozotocin-induced diabetic mice, l-leucine injection (360 μmol/100 g body weight) increased the FFR in the liver but not in muscle, while the injection of insulin (0.5 unit/100 g body weight) raised the rate in muscle but not in the liver. The leucine-induced rise of FFR in the liver was blocked by a cyclooxygenase inhibitor, indomethacin, although this inhibitor did not block the insulin-stimulated rise of FFR in muscle. No significant increase in FFR was detected in the liver or muscle of non-diabetic mice by leucine injection. l-leucine injection caused a slight decrease of ribosome aggregation in the liver but not in muscle, while insulin injection led to ribosome aggregation in muscle but not in the liver of diabetic mice. The enhancement of FFR in the liver of diabetic animals by leucine injection suggests that leucine may stimulate protein synthesis in part by increasing the rate of elongation.

Active Translation of the D-1 Protein of Photosystem II in Senescing Leaves

Primary leaves of bean (Phaseolus vulgaris L.) show pronounced symptoms of senescence within 21 days of planting. Total RNA levels decline by approximately fourfold, and pulse-labeling studies with [(35)S]methionine have indicated that synthesis of all thylakoid proteins except the D-1 protein of photosystem II is sharply curtailed. Measurements of transcript levels for D-1, which is encoded by psbA, and for two other thylakoid proteins, the 68- to 70-kilodalton protein of photosystem I encoded by psaA/B and the beta subunit of ATPase encoded by atpB/E, have indicated that the continued strong synthesis of D-1 relative to other proteins in senescing thylakoids does not reflect differential changes in message abundance. Specifically, although transcript quantity for each of these proteins decreases by approximately fourfold with advancing senescence, only the 68- to 70-kilodalton protein of photosystem I and the beta-subunit of ATPase show decreased synthesis. As well, the proportion of total psbA transcript associated with polysomes is higher in senescent leaves than in young leaves, whereas for each of psaA/B and atpB/E, there is less message in polysome formation in the senescent leaves than in young leaves. Thus, the continued strong synthesis of D-1 in senescent leaves appears to reflect preferential sequestering of ribosomes by psbA transcript from a dwindling ribosome pool. This ability to preferentially sequester ribosomes may be related to the unusually high turnover rate that characterizes D-1.

Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli

Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two-dimensional gel electrophoresis patterns. The plasmids investigated had very similar sequences except for small domains encoding the repressor of plasmid replication. At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased. At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed. Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation. Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses. These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.

Effects of dexamethasone on lung protein turnover

Dexamethasone (2.5 mg/day per kg) treatment of young growing rats resulted in reduced food intake and rapidly inhibited whole-body and lung growth. Although the reduction in food intake partially explained the decrease in whole-body growth, it did not influence lung growth. After 24 h of dexamethasone treatment, ribosomal efficiency in the lung was reduced 44%, producing a 38% decrease in the rate of pulmonary protein synthesis. Extending dexamethasone treatment to 5 days resulted in decreases in both ribosomal efficiency (35%) and capacity (28%), explaining the 53% reduction in lung protein synthesis at this time. After both the acute and chronic steroid regimes, the decreased rates of pulmonary protein synthesis were accompanied by a loss of polyribosomes and an elevated ribosomal monomer pool, indicating that dexamethasone blocked translation at the site of peptide-chain initiation.

Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post‐transcriptional events

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains. The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions. It is compatible with the previously observed effects of box A in antitermination.

Effect of undernutrition on developing rat cerebellum: Some electrophysiological and neuromorphological correlates

Electrophysiological studies of Purkinje cells from the developing normal and undernourished rat cerebellar cortex were correlated with the neuromorphological studies. In undernourished animals the firing pattern of Purkinje cells was simpler as compared to complex electrical activity in normal animals. The firing frequency of Purkinje cells remained reduced in all the age-matched undernourished groups. The duration of a spike was also prolonged as compared to their normal age mates. Similarly the mean amplitude was also relatively smaller in the undernourished animals when compared with the normal litter mates. Undernourished animals exhibited typical increased stimulus thresholds, prolonged N-2 peak wave latencies, reduced number of functional components, amplitude and increased duration of mossy fiber response (MFR). The mossy fiber (MF) activity exhibited a sluggish rise in its amplitude, when stimulus intensity was increased in undernourished animals. A phenomenon of fatigue was observed with a significant reduction in the amplitude and frequency of Purkinje cell unit discharge on repeated MF stimulation in the undernourished animals. However, repeated MF stimulation provided a high frequency discharge in the normal as well as undernourished adult rats. Neuromorphological studies at light-microscopic level exhibited delayed cell migration from the external granular layer in the undernourished rats. At electron-microscopic level, intracellular morphology exhibited almost similar ultrastructural details except for a few minor alterations. A free ribosomal pool, immature development of E.R., increased incidence of lysosomes and electron-dense membrane stacks were observed in the Purkinje cells at 30 days in the undernourished animals. The results of the present study indicated that undernutrition affects the maturation of Purkinje cell regional neurocircuitry.

Separate ribosomal pools in sea urchin embryos: ammonia activates a movement between pools.

Monoribosomes from unfertilized eggs of Strongylocentrotus purpuratus were shown to translate mRNA less efficiently than ribosomes derived from polyribosomes of embryos, as measured by globin synthesis in a ribosome-dependent rabbit reticulocyte lysate [Danilchik, M. V., & Hille, M. B. (1981) Dev. Biol. 84, 291-298]. Data presented in this paper show that monoribosomes from 16-cell and blastula embryos resemble monoribosomes from unfertilized eggs in translational capacity and are less active than the ribosomes associated with polyribosomes. Thus, we find two distinct populations of ribosomes in embryos. We define the less active monoribosome population as "naive" ribosomes and the more active, functioning polysome-derived ribosomes as "experienced" ribosomes. Naive and experienced ribosomes have the same elongation rates. The relationship between ionic triggers and the conversion of monoribosomes to experienced ribosomes was studied with the Ca2+ ionophore A23187, which releases intracellular Ca2+ stores, and NH4Cl, which alkalinizes the cytoplasm. We found that ribosomes in the monoribosome populations from A23187-activated eggs or from NH4Cl-activated eggs resembled naive monoribosomes from unfertilized eggs in their translational activity. In contrast, ribosomes derived from the polysomes of NH4Cl-treated eggs were as active as the experienced polysome-derived ribosomes from normal embryos. Eggs activated with A23187 did not produce polyribosomes. The presence of significant amounts of experienced ribosomes in NH4Cl-treated eggs implicates alkalinization of the cytoplasm as a stimulus for ribosome activation, which occurs slowly during initial development.

Distribution of hepatic ribosomes between different functional states in well-fed and protein-energy-deficient rats.

Electron microscopy of hepatic cells from protein-deficient young rats revealed extensive breakdown of the rough endoplasmic reticulum into fragments and vesicles whose membranes appeared fully studded with ribosomes. Additionally, there was biochemical evidence of marked disaggregation of polysomes (n greater than 2), and this was more prominent in the membrane-bound than in free polysomes. Protein malnutrition increased the proportion of membrane-bound ribosomes which were salt-releasable and therefore temporarily non-functional. It has been concluded that disaggregation of membrane-bound polysomes induced by malnutrition does not necessarily imply detachment of the monomeric ribosomes from the endoplasmic membrane into the pool of free ribosomes, which probably has a separate polyribosomal cycle.

Ribosome changes induced by irradiation of isolated rat liver in vitro.

Changes produced in ribosome populations by gamma irradiation of isolated livers (rat) are reported. The main effect was the increase in free ribosomes due to the liberation of membrane bound ribosomes. The radiation apparently injures the binding-site of the large ribosomal subunit, detaching its structural proteins. The disassembled ribosomes are rapidly catabolized by cell RNase. The amount in total and free ribosomes of the livers as a function of the radiation dose was assessed. The relative insensitivity to radiation of the free ribosome pool is discussed in terms of its implications for repair and transformation of the cells.

Inhibition of cellular protein synthesis by vaccinia virus surface tubules

The infection of HEp-2 cells with vaccinia virus results in a rapid and selective inhibition of cellular protein synthesis. This effect appears to be due to a structural component(s) of the infecting virion. Experiments investigating the nature of the inhibitory principle showed that a vaccinia virion component, the surface tubule (ST), inhibits protein synthesis in HEp-2 cells without affecting either RNA or DNA synthesis. Furthermore, ST added to a rabbit reticulocyte cell-free system inhibited the incorporation of radiolabeled amino acids into acid-insoluble protein. ST inhibitory activity was destroyed by heat (56° for 60 min) and by prior incubation with specific anti-ST serum. A decrease in the polyribosome complement of cells exposed to purified ST, with a concomitant increase in the free ribosome pool, suggests that the main effect of tubules on cellular protein synthesis occurs at the level of polypeptide chain initiation.

Protein synthesis on membrane-bound and free ribosomes in growing yeast

1. A method for the study of membrane-binding of ribosomes and the rate of synthesis of protein on free and bound ribosomes has been examined critically. ‘Brij 58’ has been found to be more suitable for detaching ribosomes from membranes than ‘Triton X-100’ or sodium deoxycholate since it does not alter the structure or biological activity of the ribosomes. Triton and deoxycholate remove amino acid- and ribonucleotide-containing material from ribosomes, thus decreasing their activity during protein synthesis in a cell free system. 2. The rate of protein synthesis on membrane-bound and free ribosomes, the total amount of ribosomes, and the percentage distribution of free and membrane-bound ribosomes were determined in a culture of synchronously growing yeast cells. The results show that in all probability protein synthesis in yeast cells occurs mainly on membrane-bound ribosomes. Free ribosomes are considered here to be a ribosome pool which allows the cell to increase its rate of protein synthesis rapidly. Membrane-bound and free ribosomes are viewed as different functional states, one being a state of protein synthesis and the other a latent state.

Free fibosomes and growth stimulation in human peripheral lymphocytes: activation of free ribisomes as an essential event in growth induction.

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.

Polyamines and protein synthesis: Studies in various polyamine-requiring mutants ofEscherichia coli

Different Escherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. The in vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutans MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.

Synthesis and soluble pools of ribosomal proteins in Rhodopseudomonas palustris

The specific antibodies prepared against two purified ribosomal proteins (19 and 24) and total 66-S ribosomal proteins have been utilized to measure free ribosomal proteins in Rhodopseudomonas palustris. The free ribosomal protein pool (66 S) amounts to 1–2% of the total soluble proteins of R. palustris. In addition, the size of free ribosomal protein pool (66 S) is calculated to be approx. 7% of the total ribosomal protein on the mature 66-S ribosomes from the pulse-labelling data. A study of the pool size indicates that ribosomal proteins are synthesized at a constant rate during exponential growth. However, during abnormal conditions such as antibiotic treatment, individual ribosomal proteins behave in a manner distinct from the average behavior of total 66-S ribosomal proteins. The co-ordination in the biosynthesis of free ribosomal proteins is no longer apparent during chloramphenicol treatment. Measurements on the free ribosomal protein pools following physiological stress treatment indicate that duing the recovery period, rate of ribosome assembly is greater than the rate of ribosomal protein synthesis.

Free ribosomes in physiologically nondividing cells. Human peripheral lymphocytes.

Ribosomes of physiologically nondividing human peripheral blood lymphocytes were studied by ultraviolet absorbance measurements of cytoplasmic extracts subjected to ultracentrifugation in sucrose gradients at high and low ionic strength. At least 70% of the total cytoplasmic ribosomes were free ribosomes which sedimented at 80 S in low salt and dissociated to 40 S and 60 S subunits in high salt. These particles were not involved in protein synthesis. The remaining ribosomes were equally divided among native subunits, active monosomes, and larger polysomes. Free ribosomes were shown to exist as 80 S particles in the intact cell, and labeling studies indicated that they did not freely return to the pools of protein-synthesizing components. New ribosomes appeared first as native subunits and in polysomes. After a lag of 15 to 20 min, the particles began to enter the free ribosome pool. Thus, free ribosomes arise in the resting cell by a unidirectional flow which continuously removes particles from the protein-synthesizing pool and sequesters them as an accumulation of inactive 80 S particles. The transition from native subunits to free ribosomes is accompanied by functional changes in association behavior of subunits and by alteration of sedimentation behavior of the subunits. These changes may be due to absence of a protein or proteins from the free ribosomes which is required to permit effective dissociation of subunits prior to initiation of translation. Deficiency of this dissociation factor may be responsible for the continuous formation of free ribosomes in resting cells. Our data also suggest a limitation of the rate of initiation of protein synthesis which may result from deficiency of an initiation factor.

Extracellular ribosomes during metamorphosis of the blowfly [formula omitted] R. D. -- A reappraisal of their authenticity

Extracellular ribosomes during adult development of the blowfly Calliphora vicina were previously considered to occur naturally in vivo . A variety of radioisotopic experiments performed at different stages of metamorphosis now demonstrates that the specific activities of extra- and intracellular ribosomes are consistently very similar. Further, in addition to previously described physico-chemical similarities, extra- and intra-cellular ribosomal protein profiles are now shown to be essentially identical. These results vitiate the notion of a natural pool of extracellular ribosomes, the occurrence of which is now ascribed to an experimental artifact, resulting from unusual cell fragility.