Nutritional Shift-up Research Articles

Balanced production of 30S and 50S ribosomal proteins after a nutritional shift-up.

The synthesis of bulk ribosomal protein (r-protein) after a nutritional shift-up in Escherichia coli B/r was examined. It was found that the molar ratio of the net synthesis rates of 30S and 50S r-protein remains constant during the transition period after the shift-up and equal to the preshift ratio. The implications for the control of ribosome synthesis are discussed.

Protein synthesis during a nutritional shift-up in [formula omitted

The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in Escherichia coli ; while the rate of synthesis of elongation factors did not increase until 5–10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron.

Regulation of the expression of ribosomal protein genes in Escherichia coli

The in vivo expression of genes coding for ribosomal proteins has been examined by using a quantitative RNA-DNA hybridization assay. Total cellular RNA isolated from exponential-phase cultures of Escherichia coli growing at rates between 0·67 and 2·10 doublings per hour and from cultures following a nutritional shift-up was hybridized to DNA from a λ transducing phage carrying at least 22 ribosomal protein genes. These r-protein genes code for about 400,000 daltons of protein or about half of the total protein per ribosome. The results indicate that the fraction of the total cellular RNA which is homologous to the r-protein genetic region on the transducing phage, s , increases with increasing growth rate such that s = 0·0016 μ + 0·0006, where μ is the steady-state growth rate in doublings per hour. Comparison of the rate of synthesis of the r-proteins coded for by genes on the phage and the level of r-protein messenger RNA homologous to these genes during steady-state and transition conditions indicates that the expression of r-protein genes is controlled primarily by a mechanism which regulates the amount of mRNA for r-protein which is available for translation. This control may be exercised either at the level of initiation of transcription at the promotors for r-protein genes or at the level of the stability of the r-protein mRNA. The results further suggest the existence of a second control mechanism which influences the translation of r-protein mRNA.

Transition period following a nutritional shift-up in the bacterium Escherichia coli B/r : Stable RNA and protein synthesis

The previously observed patterns of stable RNA and protein synthesis during the transition period following a nutritional shift-up are analyzed and described by kinetic equations. (i) The kinetics of the stable RNA synthesis rate after a shift-up are diphasic; the first phase, a step-like increase occurring within minutes after the shift, reflects an induction of rRNA and tRNA transcription; the second phase, an exponential increase with a higher (post-shift) rate constant, reflects a change in the accumulation rate of functioning RNA polymerase. The ratio of the numbers of ribosomes and functioning RNA polymerase molecules is shown to be independent of growth rate or time after the shift. (ii) The protein synthesis rate increases exponentially throughout the transition period with a rate constant corresponding to that of post-shift steady-state growth, which reflects the exponentially increasing number of ribosomes. An initial step-like increase in the protein synthesis rate due to an increase in the ribosome efficiency occurs when the pre-shift growth is below 1·2 doublings/hr. (iii) The duration of the transition period is estimated from the time it takes for the stable RNA to protein ratio to reach its post-shift steady-state value. The period is approximately 3 hr but has no clearly defined end. (iv) When RNA or protein synthesis rates are measured in different units, for example as rate per unit of culture volume, rate per cell or per genome, these differently defined rates change in different patterns after a shift-up. The relationships between these units and the resulting kinetics are explained.

Control of cell length in Bacillus subtilis.

During inhibition of deoxyribonucleic acid synthesis in Bacillus subtilis 168 Thy-minus Tryp-minus, the rate of length extension is constant. A nutritional shift-up during thymine starvation causes an acceleration in the linear rate of length extension. During a nutritional shift-up in the presence of thymine, the rate of length extension gradually increases, reaching a new steady state at about 50 min before the new steady-state rate of cell division is reached. The steady-state rates of nuclear division and length extension are reached at approximately the same time. The ratio of average cell length to numbers of nuclei per cell in exponential cultures is constant over a fourfold range of growth rates. These observations are consistent with: (i) surface growth zones which operate at a constant rate of length extension under any one growth condition, but which operate at an absolute rate proportional to the growth rate of the culture, (ii) a doubling in number of growth zones at nuclear segregation, and (iii) a requirement for deoxyribonucleic acid replication for the doubling in a number of sites.

Metabolic regulation of β-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis

The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.

Regulatory state of ribosomal genes and physiological changes in the concentration of free ribonucleic acid polymerase in Escherichia coli.

The concept of promoter efficiency is introduced as frequency of RNA chain initiation at a given promoter normalized to the intracellular concentration of free (but functional) RNA polymerase. Previous observations from this laboratory on the synthesis of ribosomes and beta-galactosidase are used to show that during a nutritional shift-up from succinate minimal to glucose-amino acids medium (3-fold increase in steady-state growth rate) the concentration of free (active) RNA polymerase decreases to one-quarter of the pre-shift value and the promoter efficiencies of the genes for ribosomal RNA and ribosomal proteins increase 9- and 6-fold respectively. This extent of control of ribosomal genes is much greater than expected on the basis of the increase in the rate of ribosome synthesis (3-fold).

Replication of R-factor R1 in Scherichia coli K-12 at different growth rates

The R-factor R1drd-19 mediates resistance to beta-lactam antibiotics via a beta-lactamase. A strain of Escherichia coli K-12 carrying R1drd-19 was grown at different growth rates by using different carbon sources. The specific rate of production of the R1 beta-lactamase increased linearly with the growth rate and with the gene dosage. The content of R1 deoxyribonucleic acid was estimated by alkaline sucrose gradient centrifugation and by analysis of the specific rate of beta-lactamase synthesis in nutritional shift-up experiments and was found to decrease fivefold when the growth rate was increased from 0.4 to 1.8 doublings per h. The number of R1 molecules per cell decreased from six to two in the same growth range. The presence of the plasmid affected the mean cell size significantly; at a growth rate of 0.4 doublings per h the R-+ cells were on the average 50% bigger than the R-minus cells, whereas the effect was less than 10% at a growth rate of 1.8 doublings per h. Several reports in the leterature state that the initiation mass of chromosome replication is constant. In this paper it is shown that the initiation mass of R1 replication is proportional to the growth rate. Thus, the replication of the plasmid R1 and of the chromosome are independently regulated processes. It is argued that plasmid replication is under negative control.

Synthesis and turnover of basal level guanosine tetraphosphate in Escherichia coli.

Cultures of escherichia coli growing exponentially in Trisacetate medium were subjected to nutritional shift-up and the pool size of guanosine 5'-3'-diphosphate-3'diphosphate (ppGpp) as well as the rates of protein synthesis and net RNA synthesis were determined. In the shift to a rich medium (glucose plus 19 amino acids plus hypoxanthine) the basal level of ppGpp falls immediately with a decay constant suggesting total inhibition of synthesis; ther is no ppGpp detectable above background for 30 to 40 min. The net rate of RNA synthesis starts to increase within 1 min of the shift-up and has reached its definite postshift value well before the pool of ppGGpp rises above background lvel. In a shift-up from Tris-acetate medium to Tris-glucose medium there is a much slower readjustment of the ppGpp pool size without the transient disappearance of the nucleotide. However, in a shift-up to Tris-acetate plus 5 amino acids, a medium which supports the same growth rate as Tris-glucose medium, a dramatic, transient drop in the ppGpp pool level was observed. Relaxed cells exhibit very similar behavior to strigent cells in the same shift-up. Our data argue strongly against an exclusive role for pGpp in regulating RNA synthesis during niutritional shift-up. The kinetic data of [3H]guanosine uptake into GTP and ppGpp pools were analyzed to determine the rate of pGpp synthesis. This rate was found to be similar during expotential growth in either Tris-acetate medium. During a shift-down from Tris-glucose to Tris-acetate medium the rate of ppGpp syntesis fell by a factor of 1.5 to 2 and the turnover rate is reduced 6- to 8-fold, suggesting that the expansion in the ppGp pool during shift-down canot be due to derepression of synthesis.

Regulation of ribonucleic acid synthesis in Escherichia coli B/r: An analysis of a shift-up : III. Stable RNA synthesis rate and ribosomal RNA chain growth rate following a shift-up

Following a nutritional shift-up, both the fraction of functioning RNA polymerase engaged in the synthesis of stable RNA, ψ s, and the ribosomal RNA chain growth rate, c s, increase within five minutes to near their final post-shift steady-state values. The increase in these two parameters is sufficient to account completely for the observed sudden increase in the rate of RNA accumulation. This implies that the control of stable RNA synthesis following a shift-up does not involve an activation of an inactive reserve of RNA polymerase or a burst of RNA polymerase synthesis, but rather results from a shift of RNA polymerase-transcribing messenger RNA genes to ribosomal and transfer RNA genes along with some increase in the stable RNA chain growth rate.

Synthesis of individual ribosomal proteins in Escherichia coli B/r

The differential synthesis rates of individual ribosomal proteins (r-proteins) were measured in Escherichia coli B/r during the transition period following a nutritional shift-up from succinate minimal to glucose/ammo acids medium. These rates were observed to respond sequentially to the shift-up; the differential synthesis rate of protein L28 begins to increase within 0.1 of a minute following the shift-up, while the protein L29 synthesis rate begins to increase only after a lag of 2.5 minutes. The onset of induction of the remaining r-proteins occurs within this 2.5-minute interval. Furthermore, there was a twofold variation in the acceleration of the differential synthesis rates of individual r-proteins. Within the initial two to ten-minute period following the shift-up the differential synthesis rates of most r-proteins reached values ranging from 2.2 to 3.0-fold higher than the pre-shift rates, before declining to the post-shift steady-state values. It is suggested that the increases in the differential synthesis rates of r-proteins result in part from increases in the translational efficiency of messenger RNA in the post-shift growth medium and in part from increases in the amount of r-protein mRNA that is present.

The size of transcriptional units for ribosomal proteins in Escherichia coli. Rates of synthesis of ribosomal proteins during a nuritional shift-up.

The kinetics of the rate of synthesis of the ribosomal proteins has been determined during a nutritional shift-up in E. coli. Evidence is obtained for a maximal transcription time of 2 min for possible transcriptional units for ribosomal proteins. This is in accordance with the earlier conclusion that ribosomal protein genes are clustered in several relatively small transcriptional units (Molin et al., 1974).

Unused protein synthetic capacity of Escherichia coli grown in phosphate-limited chemostats

Escherichia coli was grown in phosphate-limited chemostat cultures at dilution rates corresponding to 8 to 12-hour doubling times. The RNA content expected in such cultures based on the “constant efficiency” hypothesis was six to eight-fold lower than that actually found. Measurement of the growth kinetics, pulse incorporation of guanine and tryptophan following nutritional shift-up, and the determination of delay time for the completion of the first polypeptide of β -galactosidase after addition of inducer lead to the following conclusions. (1) The “extra” RNA corresponds to unused protein synthetic capacity that is not in use in phosphate-limited growth. (2) However, those ribosomes actually engaged in protein synthesis do operate as efficiently as in faster growing cells. (3) Immediately (less than five minutes) on being shifted-up, the previously unused protein synthetic capacity does function. (4) The rate of RNA accumulation after a nutritional shift-up from phosphate-limited growth accelerates quickly, then decelerates, and then accelerates more slowly to the steady-state rate.

Synthesis of individual ribosomal proteins during a nutritional shift-up.

Although individual ribosomal proteins are synthesized at the same rate during exponential growth, during a nutritional shift‐up, they are synthesized at different rates. Proteins S6, S10, S14, S20 and L2, L3, L4 and L13 are synthesized to the greatest extent. Proteins S2, S3, S4, S13, S17 and L7, L8, L9, L25, L29 and L30 are synthesized at the lowest rate. Although the average rate of synthesis of 50‐S protein is greater than that for 30‐S protein during a shift‐up, the data on rates of synthesis of individual proteins do not support such a distinction for each individual ribosomal protein.The extent of increased synthesis for the individual ribosomal proteins of the 30‐S subunits correlated well with the assembly order of these proteins. This correlation is not as striking for the 50‐S subunit.

Differential rate of ribosomal protein synthesis in Escherichia coli B/r

The differential rate of ribosomal protein synthesis, α r (ribosomal protein synthesis rate/total protein synthesis rate), was measured for Escherichia coli strain B/r growing at different steady-state rates ranging from 0.67 to 2.3 doublings/hour. For growth rates above 1.2 doublings/hour, α r was found to be proportional to the growth rate μ (doublings/h), such that α r = 0.09 μ, and the ribosome efficiency (amino acids polymerized/second per ribosome), calculated from α r, was found to be 14 to 18 amino acids/second per ribosome. With decreasing growth rates below 1.2 doublings/hour, α r was found to be increasingly greater than 0.09 μ and the ribosome efficiency gradually decreased such that at μ = 0.67, α r = 0.085, and the ribosome efficiency was reduced by 30% and was equal to 10 to 13 ammo acids/second per ribosome. These results imply that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 × 10 8 to 5 × 10 8 amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10 8 to 4 × 10 8 amino acids/genome. These relationships were verified by direct measurements of the amounts of DNA, RNA and protein at different steady-state growth rates. In addition, protein accumulation was measured following a nutritional shift-up from succinate to glucose minimal medium. The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up.

Activity of polypeptide chain initiation factors during a nutritional shift-up in Escherichiacoli

Summary During the first 10–15 minutes following nutritional shift-up in Escherichia coli , no significant increase was detected in the specific activity of initiation factors. The next 25 minutes, however, were characterized by a doubling of specific activity of these factors. Measurements of initiation factor activity of different exponential growth rates revealed a linear proportionality between growth rate and initiation factor activity. For each doubling of growth rate, initiation factor activity increased 1.6–1.8 fold.

Induction of nucleolar and mitochondrial DNA replication in Tetrahymena pyriformis.

Selective induction of replication of nucleolar and mitochondrial DNA has been demonstrated in starved-refed cultures of Tetrahymena pyriformis by different techniques. Labeling of starved cells with [(3)H]thymidine during a nutritional shift-up and analysis of the DNA in isopycnic CsCl gradients shows that the two initially labeled species of DNA are two species banding on the heavy and light side of the bulk macronuclear DNA. In isolated macronuclei the radioactivity is found only in the high density fraction, which has been shown to be of nucleolar origin. In sucrose gradients the newly replicated mitochondrial and nucleolar DNAs sediment considerably slower than the bulk DNA, as one discrete band corresponding to a molecular weight of about 3 to 4 x 10(7). Electron microscope autoradiography of cells labeled with [(3)H]thymidine as above shows that the peripheral nucleoli of the macronucleus as well as the mitochondria are labeled before any radioactivity is found in the chromatin granules of the macronucleus. The results clearly indicate that nucleolar and mitochondrial DNA replication are under a control independent of that for the replication of bulk DNA.

Ribosome production and elongation factor activity during a nutritional shift-up in Escherichia coli

Ribosome production and elongation factor activity during a nutritional shift-up in Escherichia coli

Coupling between chromosome completion and cell division in Escherichia coli.

The relationship between termination of chromosome replication and cell division was investigated in Escherichia coli B/r. Synchronous cultures of E. coli B/r growing in glucose minimal medium or subjected to a nutritional shift-up were exposed to chloramphenicol, rifampin, mitomycin C, or nalidixic acid, and the ability of cells to divide in the presence of the inhibitors was measured. It was found that cell division became resistant to inhibitors of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis at approximately the same stage in the division cycle in all situations investigated. When the synchronous glucose-grown cultures were temporarily exposed to chloramphenicol early in the division cycle and then exposed to mitomycin C or nalidixic acid immediately after removal of chloramphenicol, the cells did not divide. In contrast, when DNA synthesis was inhibited by thymine starvation immediately after temporary exposure to chloramphenicol, cells divided. The results suggest that DNA chain elongation is completed in some cells in the absence of protein synthesis, but that additional steps involving specific RNA or protein synthesis, or both, may be required for processing the chromosomal structures to the form which is necessary for division. This processing, which normally occurs concurrent with DNA synthesis and is prevented by inhibitors of DNA synthesis, may trigger division. Alternatively, in the absence of protein synthesis, all aspects of chromosome formation may be completed, but final transcriptional events which are essential for division cannot take place until the complete synthesis of a critical amount of specific proteins.

Cell division and deoxyribonucleic acid synthesis after a nutritional shift-up of Saccharomyces cerevisiae.

Rates of cell division and deoxyribonucleic acid synthesis after shift-up with grande and mitchondrial deoxyribonucleic-acid-less petite yeasts were studied. The results indicate that simple eukaryotes behave as prokaryotes.