Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate.

By evaluating the kinetics of radioactive labelling of nascent and finished polypeptides, the peptide-chain elongation rate for Escherichia coli B/r at three different growth rates (mu) was determined to be 17 amino acids/s for the fast-growing cells (mu equals 1.3 and 2.0 doublings/h) and 12 amino acids/s for slow-growing cells (mu equals 0.67 doublings/h). The results agree with the growth-rate-dependence of the rate of peptide-chain elongation found for the translation of newly induced beta-galactosidase messenger in this strain and under these conditions of growth [Dalbow & Young (1975) Biochem. J. 150, 13-20]. Together with the previously observed ribosome efficiency at these growth rates [Dennis & Bremer (1974) J. Mol. Biol. 84, 407-422] the results indicate that the fraction of ribosomes engaged in protein synthesis is about 0.8 at all three growth rates.     

The concentrations of stable RNA and ribosomes in Rickettsia prowazekii

The obligate Intracellular parasite, Rickettsia prowazekii, is a slow-growing bacterium with a doubling time of about 10h. In the present study, DNA and RNA were obtained from the rickettsiae by two independent methods, i.e. simultaneous isolation of DNA and RNA from the same sample by phenol:chloroform extraction and CsCI gradient centrifugation. In addition, ribosomal RNA was obtained by sedimentation of partially purified ribosomes from the rickettsiae. The results demonstrated that, after correction for the cell volumes, the concentrations of stable RNA and ribosomes in R prowazekii, a slow-growing organism, were about 62fg μm⁻³ and 17000 per μm³, respectively, which were very simitar (66fg μm⁻³ and 21 000 per μm³) to those in Escherichia coli with a generation time of 40min. However, on a per cell basis, R. prowazekii had 5.6 fg of RNA and 1500 ribosomes per cell, which was only about 8% of the amount of both stable RNA (71.2 fg) and ribosomes (24000) per cell as was found in E. coli. These results indicated that R. prowazekii possesses a ribosome concentration greater than might have been predicted from its slow growth rate. This high concentration of ribosomes could be due to a large population of non-functioning ribosomes, a low efficiency of amino acid production, or a high rate of protein turnover. However, this study also demonstrated that the rickettsiae have very limited protein turnover. Knowledge of the kinetics and control mechanisms for protein synthesis in R. prowazekii remains to be established to determine the logic of the extra rickettsial ribosomes.     

Translational Control of Protein Synthesis in Staphylococcus aureus.

The calculated in vivo polypeptide chain growth rate for Staphylococcus auteus MF-31 grown in nutritionally rich medium assuming all the ribosomes were functional was found to be approximately 16 amino acids/s/ribosome, but decreased to 10.2 amino acids/s/ribosome for cells grown in poor medium. An in vitro analysis revealed that 70S ribosomes isolated from rich medium cells were more active than similar 70S ribosomes derived from cells grown in poor medium. The 30S subunit was found responsible for the increased activity of the rich monosomes, whereas the 50S subunit appeared to be capable of either high or low activity.     

Ribosome biosynthesis in Tetrahymena pyriformis. Regulation in response to nutritional changes

Ribosome contents of growing and 12-h-starved Tetrahymena pyriformis (strain B) were compared. These studies indicate that (a) starved cells contain 74% of the ribosomes found in growing cells, (b) growing cells devote 20% of their protein synthetic activity to ribosomal protein production, and (c) less than 3% of the protein synthesized in starved cells is ribosomal protein. Ribosome metabolism was also studied in starved cells which had been refed. For the first 1.5 h after refeeding, there is no change in ribosome number per cell. Between 1.5 and 2 h, there is an abrupt increase in rate of ribosome accumulation but little change in rate of cell division. By 3.5 h, the number of ribosomes per cell has increased to that found in growing cells. At this time, the culture begins to grow exponentially at a normal rate. During the first 2 h after refeeding, cells devote 30-40% of their protein synthetic activity to ribosomal protein production. We estimate that the rate of ribosomal protein synthesis per cell increases at least 80-fold during the first 1-1.5 h after refeeding, reaching the level found in exponentially growing cells. This occurs before any detectable change in ribosome number per cell. The transit time for the incorporation of these newly synthesized proteins into ribosomes is from 1 to 2 h during early refeeding, whereas in exponentially growing cells it is less than 30 min. The relationship between ribosomal protein synthesis and ribosome accumulation is discussed.     

Ribosome Production and Protein Synthesis in the Preimplantation Rabbit Embryo

Biochemical and radioautographic data show that protein synthesis is increased markedly at the morula stage of rabbit development (60 h embryo). In the late morula an increase in cytoplasmic ribosomes is observed, suggesting that ribosome availability may be rate-limiting for protein synthesis during cleavage. Incorporated ³H-amino acids become highly localized within the nucleoli of late morulae which have been pulse-labelled for 10 min. This localization suggests that ribosomal protein synthesis is increased at the same time as ribosomal RNA synthesis has been shown to increase. Changes in both the incorporation of ³H-amino acids and cytoplasmic ribosome density were found to occur 'synchronously' in all embryonic cells during the cleavage and early blastocyst period (84 h of development). Between 84 h and 108 h, considerable differences in the number of ribosomes per unit area of cytoplasm become apparent among the cells of the blastocyst.     

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 × 10⁸ to 5 × 10⁸ amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10⁸ to 4 × 10⁸ 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.     

Synthesis time of beta-galactosidase in Escherichia coli B/r as a function of growth rate.

By analysing the kinetics of beta-galactosidase accumulation after induction, the synthesis time of beta-galactosidase in Escherichia coli B/r was found to be 75s in rapidly growing cells (1.36 and 2.1 doublings/h), and 90s in slowly growing cells (0.63 doubling/h). These values correspond to peptide-chain-elongation rates of 16 and 13 amino acids/s respectively, in agreement with previous findings, indicating that the peptide-chain growth rate is constant (presumably maximal) in fast-growing bacteria, but decreased in slowly growing bacteria [Forchhammer & Lindahl (1971) J. Mol. Biol. 55, 563-568].     

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

Summary Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: -N-trimethyllysine, -N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.     

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.     

The inefficiency of ribosomes functioning in Escherichia coli growing at moderate rates

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

Changes in ribosomes associated with spore senescence in the bean rust fungus.

Studies were carried out to idenify the cause of the decline in transferase activity and capacity to bind polyuridylic acid which occurs in ribosomes from germinated uredospores of the bean rust fungus, Uromyces phaseoli (Pers.) Wint., aged longer than 6 h on a water surface. We have shown that such ribosomes lose the capacity to respond to added transferase-I and that both subunits were affected by the ageing process. These changes were not accompanied by a significant alteration in the composition of the ribosome. However, deoxycholate had a greater detergent effect on ribosomes from germinated spores than from nongerminated spores as shown both by loss of capacity to polymerize amino acids and loss of protein. Ribonuclease activity did not increase during germination, but the amount found (Imug/g spores) was easily detectable. It was suggested that loss of response to transferase-I was due to an alteration of ribosomal proteins of both subunits.     

Import of frog prepropeptide GLa into microsomes requires ATP but does not involve docking protein or ribosomes.

Frog prepropeptide GLa, a precursor to a secretory protein containing 64 amino acids, was processed and imported by dog pancreas microsomes. These events did not depend on either docking protein or on the presence of ribosomes. A hybrid protein between the first 60 amino acids of prepropeptide GLa and an unrelated peptide of 49 amino acids fused to the carboxy terminus, however, behaved like a typical secretory protein precursor with regard to docking protein dependence. This suggests that independence of the need for docking protein, in the case of prepropeptide GLa, can be attributed to the size of the precursor protein. Processing and import of prepropeptide GLa by microsomes were ATP dependent. Therefore, import of proteins into the endoplasmic reticulum (ER) includes an ATP-requiring step not involving a ribosome/ribosome receptor or signal recognition particle (SRP)/docking protein interaction.     

Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro

Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.     

SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION : Production of Eggshell Proteins by Silkmoth Follicular Cells

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.     

Generation of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: evidence that ribosome depurination is not sufficient for cytotoxicity

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved α-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5′ and the 3′ ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.     

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.