Number Of Active Ribosomes Research Articles

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h−1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

A counting-strategy together with a spatial structured model describes RNA polymerase and ribosome availability in Escherichia coli

The allocation of resources during bacterial growth is strongly related to the availability of ribosomes and RNA polymerase molecules. Here, coarse-grained models offer a promising start due to their simple structure and the limited number of kinetic parameters. Based on published data sets for proteome and mRNA data in Escherichia coli, and together with mass balance equations describing gene expression, we are able to calculate the number of active molecules (that is, the number of ribosomes that are currently translating nascent and mature mRNA, as well as the number of RNA polymerase molecules on the DNA). This information is a prerequisite for meaningful coarse-grained models. In our approach, the cellular compartment is structured into a cytosolic region and a nucleoid region, and the processes of transcription and translation are assigned accordingly. The theoretical study reveals a quadratic relationship between the number of active ribosomes and the growth rate μ. While the overall available number of ribosomes follows the linear “bacterial growth law”, the approach allows us to determine the growth limit for the chosen experimental environment (minimal medium, only one C source). The new approach is in good agreement with published experimental data, and, with a simple rule of thumb can be applied to other cellular systems.

Translational initiation factor eIF-2 subcellular levels and phosphorylation status in the developing rat brain

We have quantified the levels of the α subunit of initiation factor 2 (eIF-2) in the postmicrosomal supernatant and the ribosomal salt wash fractions from suckling and adult rat brain. The levels of eIF-2 in the ribosomal salt wash decrease in adult with respect to that present in suckling rat brain, but the total amount remains fairly constant, and a very close parallelism exists between the eIF-2 associated with ribosomes and RNA levels in the microsomal fraction in the two age groups. The phosphorylation state of eIF-2α, as determined by isoelectric focusing followed by protein immunoblotting, in the same subcellular fractions, did not reveal the presence of the phosphorylated form in any of the fractions studied. These results suggest that phosphorylation of the α subunit is not implied in the regulation of protein synthesis initiation during brain development, and some other component regulates both the number of active ribosomes and eIF-2 levels in microsomes.

Effect of chronic ethanol consumption on hepatic mitochondrial transcription and translation

Liver mitochondria from ethanol-fed rats display an impaired ability for protein synthesis in vitro. Studies were conducted to explore the possible mechanisms which might account for this impaired capacity of ethanol mitochondria for protein synthesis. The present studies did not demonstrate any significant ethanol-induced lesion in mitochondrial nucleic acid metabolism in organelles isolated from ethanol-fed rats for any of the parameters investigated (mtDNA content, steady-state mtRNA concentration, mtRNA polymerase activity, concentration of specific mRNAs and rRNAs, mtRNA processing). An investigation of ribosome function in isolated mitochondria demonstrated significant decreases in the number of active ribosomes (55% fewer) in mitochondria from ethanol-fed rats. Initiation of protein synthesis was also significantly depressed (46%) in ethanol mitochondria. In addition, the yield of ribosomal particles from ethanol mitochondria was decreased 32% as compared to the yield of ribosomal particles from control mitochondria. However, isolated ribosomes from ethanol mitochondria were determined to be fully functional in a poly(U)-directed phenylalanine polymerization system. Soluble translation factors from ethanol mitochondria were also found to support full activity of control ribosomes in a poly(U)-directed phenylalanine polymerization system. These results suggest strongly that the ethanol-induced depression of mitochondrial protein synthesis is due to a decrease in the number of competent ribosomes in hepatic mitochondria from chronically ethanol-fed rats.

Heat shock, protein synthesis and ribosomal protein S6 phosphorylation in vitro in Yoshida AH 130 ascites hepatoma cells

An activated 40S ribosomal protein S6 kinase has been demonstrated previously in cytosolic extracts from proliferating as well as resting cells of a very undifferentiated rat ascites hepatoma cell line (Yoshida AH 130), grown in vivo (Cell Biol. Int. Rep., 1986, 10, 821–831). In the present report we present evidence of unmodified activity of this kinase and S6 phosphorylation in vitro in cells submitted to a physiological stress such as a sublethal temperature elevation (heat shock: 42 °C for 2 h). The heat treatment causes a progressive decline in the number of active ribosomes and of L- 35S methionine incorporation into total protein, suggesting drastically decreased synthesis of cellular proteins under these conditions. Cells recovering from heat shock show the induced synthesis of a protein with an apparent Mr of 50 kDa. Spontaneous high expression of heat shock proteins (HSP 70, 89, 100), without heat shock, occurs in these tumor cells.

Influence of Protein and RNA Synthesis Inhibitors on Methylcholanthrene-Mediated Induction of Aryl Hydrocarbon Hydroxylase Activity in Intestinal Mucosa

The sensitivity of the 3-methylcholanthrene (MC)-mediated induction of aryl hydrocarbon hydroxylase (AHH) activity to pretreatment with inhibitors of protein and RNA synthesis was studied. Cordycepin significantly inhibited the induction at 12 h but not at 3 h post-treatment with MC. Both cycloheximide and actinomycin D significantly inhibited the MC-mediated induction at 3 and 12 h after MC treatment. An MC-mediated stimulation of 3H-leucine incorporation into microsomal protein in vivo was observed at 12 h following MC treatment. A cell-free protein synthesis system employing intestinal ribosomes was developed. The rate of protein synthesis (3H-leucine incorporation/mg rRNA/3 min) and the number of active ribosomes (3H-peptidyl-puromycin formed/mg rRNA) were determined. No effect of in vivo MC treatment was observed in subsequently isolated intestinal ribosomes at either 3, 9 or 18 h post-treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on intestinal microsomes prepared from control, corn-oil- and MC-treated rats 18 h after treatment. The microsomal proteins depicted only slight quantitative changes in microsomal proteins following MC treatment, and no unique protein peaks. These results may explain the lack of detectable stimulation of the activity of isolated ribosomes. In addition, the methods used may lack sufficient sensitivity to detect the small net change in protein necessary to account for the increase in AHH activity seen after MC induction.

A decrease in the number of active mouse liver ribosomes during aging

Microsomes, deoxycholate treated polysomes, and molar KCl treated monosomes were prepared from eleven and twenty-eight month old mouse liver. The number of active ribosomes were estimated in these preparations by measuring the ability to form peptidylpuromycin under conditions in which no recycling of ribosomes occur. The results showed that the decreased synthetic capacity of preparations from the older animals [as measured by endogenous and polyphenylalanine synthesis in response to poly(U)] was most likely a result of a smaller proportion of active ribosomes rather than a reduced efficiency of individual ribosomes.

Number and activity of active ribosomes in bacterial polyribosomes.

Polyribosomes were isolated from Baccillus licheniformis, grown at 37 and 46 degrees C, and from Bacillus stearothermophilus, grown at 46 and 55 degrees C. The polyribosomes were incubated with either [3H]puromycin or [14C]phenylalanine. The number of active ribosomes (i.e. those to which growing polypeptide chains are attached) was calculated from the amount of [3H]peptidyl-puromycin formed. The activity of an active ribosome (i.e. the total number of amino acid molecules incorporated/unit time per active ribosome) was calculated from the uptake of [14C]phenylalanine. The number of active ribosomes per migrogram of RNA was as follows: for B. licheniformis, 1.66 X 10(12) and 1.72 X 10(12) at 37 and 46 degrees C respectively; for B. stearothermophilus, 2.59 X 10(12) at 46 and 55 degreesC respectively. The activity per active ribosome was as follows: for B. licheniformis, 0.61 and 0.05 at 37 and 46 degrees C respectively; for B. stearothermophilus, 0.58 and 0.42 at 46 and 55 degrees C respectively.

RNA and protein elongation rates in Saccharomyces cerevisiae.

The RNA elongation rate has been measured in yeast by the kinetics of appearance of radioactivity in the different molecular weight classes by the method first developed by Bremer and Yuan (1968). Despite the limitations caused by the breakdown of the 35s rRNA precursor, an estimate of 29 to 38 nucleotides/second at 30° has been obtained for the RNA elongation rate. The protein elongation rate has been calculated by the method of Maaloe and Kjeldgaard (1966) which consists of dividing the number of amino acids polymerized into protein per unit of time by the number of active ribosomes. This has given values of 7 to 9 amino acids/second at 30°.

Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.

Chapter 2 Steps of Realization of Genetic Information in Early Development

Publisher Summary One of the most important problems pertaining to the mechanisms of development is to find out the events that take place between the time a gene comes into operation and its phenotypic expression. In the cells of the adult organism, regulation is predominant; in cell differentiation, a program prevails. The maintenance of the constant rate of translation or enzymatic activity is realized by the mechanisms of regulation, but the regular changes in the transcription pattern or those in the rate of protein synthesis in the course of development are carried out in accordance with a program. The mechanisms ensuring the regulation and programming are, evidently, basically different, as a regulatory apparatus must respond to a systems deviation from a given parameter, whereas the program implies alteration of the parameters in time. A programming unit should, therefore, include a time-meter, for example, an indicator of distance on the road or of developmental stage in the embryo. The mechanisms of realization of the regulation and of the program, however, may be similar: a turn of the wheel in the car or a change in the number of active ribosomes in the course of protein synthesis.

Determination of the Number of Active Ribosomes in Muscle: Effect of Diabetes and Insulin

Determination of the Number of Active Ribosomes in Muscle: Effect of Diabetes and Insulin