Accuracy Of Protein Synthesis Research Articles

Why can't a cell grow infinitely fast?

Living cells are esoteric physiochemical systems that have evolved to survive and reproduce in their naturl environment. Under balanced conditions of growth, bacteria are probably systems as simple as any kind of free-living organism. Evolutionary forces, seemingly, should have driven prokaryotes to be very efficient. In part that is so; they make effective use of the machinery most expensive for the cell, i.e., the ribosomes and associated factors. But the evidence is that the efficiency with which they use the ribosomal machinery increases as the environment provides more favorable conditions for balanced growth. This article emphasizes the limitation to growth under optimal conditions. The role of fluctuations in the environment and the cost of accurate protein synthesis are discussed as reasons for the upper limit in obtainable specific growth rate.

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [ 35S]cysteine with incorporation of [ 3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1−2)·10 −3 cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3·10 −4 cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9·10 −6 cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.

Translation fidelity in the aging mammal: Studies with an accurate in vitro system on aged rats

The accuracy of poly(U) translation was measured in the post-mitochondrial supernatant from whole brain of 7- and 33-month-old Fischer 344 rats. Measurements were made: (i) under in vitro conditions in which translation fidelity was similar to what is known about the accuracy of translation in vivo; and (ii) under stresses of varying Mg 2+ concentrations (3–12 mM), pH (6.6–8.4), temperature (26–42°C) and in the presence or absence of 2.4% ethanol. No significant difference could be detected between the responses of old and young extracts, the activities of their Phe- and Leu-tRNA synthetases, and their endogenous amounts of Phe-tRNA and Leu-tRNA, despite the fact that the rats studied corresponded in age (by actuarial criteria) to 90-year-old human beings. The accuracy of poly(U) translation was also studied: (i) in liver and hippocampus extracts from 7- and 33-month-old rats; and (ii) in brain extracts from 3- and 29-month-old rats. The result were similar to those obtained in brain extracts from 7-month-old rats. Explanations are provided for the inconsistencies which exist in the literature regarding the effect of aging on the accuracy of protein synthesis. It is shown that the inconsistencies are likely to reflect inadequate methodology in three previous studies rather than biological diversity in the control of translation fidelity in aged animals.

Comparative analysis of translation accuracy in an Escherichia coli and a mammalian cell-free system

The effect of environmental stress on the accuracy of protein synthesis in an Escherichia coli and a rat brain cell-free system was investigated. Poly-U was translated in a rat brain and an E. coli cell-free extract under identical ionic conditions. The fidelity of translation, both in the E. coli and the rat brain extracts, was commensurate with what is known about the accuracy of translation in vivo. The incorporation of phenylalanine (code: UUU) and leucine (code: CUU, UUG or A) was measured at various Mg 2+ concentrations (3 to 22 m m), various pH's (6.6 to 8.6), various temperatures (23 to 42 °C), and in the presence or absence of 2.4% (v/v) ethanol. It was observed that (i) the accuracy of translation was generally higher in extracts from E. coli than from rat brain, and (ii) relative to that in E. coli, the translation fidelity in rat brain extracts was about 2 times more sensitive to ethanol, at least 5 times more sensitive to temperature, and at least 50 times more sensitive to pH. It was found that this differential sensitivity was not due to a differential behavior of the bacterial and the mammalian aminoacyl-tRNA synthetases under stress, but rather to the process of chain elongation itself. It is concluded that the accuracy of protein synthesis is more resistant to environmental stress in E. coli extracts than in extracts from at least one mammalian tissue.

Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes.

Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.

The Fidelity of Translation

Publisher Summary The chapter discusses the fidelity of translation. Several factors that influence the fidelity of in vivo and in vitro translation are discussed. The in vitro systems that are more errorprone can be improved by controlling factors, such as temperature, pH, ionic strength, energy regeneration, and composition of the system with respect to polyvalent cations, tRNAs, and protein factors. The notion that speed and accuracy of protein synthesis are opposing parameters is questioned on the basis of a large number of data indicating that fast systems are more accurate. An important factor that is relevant for accurate translation is perhaps the coordinated movement of messenger bound to several ribosomes in a polysome. Two other types of translational errors have been reported. One type of error involves a “readthrough” translation of certain termination codons in both prokaryotes and eukaryotes by a normal tRNA. The other type, demonstrated more recently, is an initiation error taking place in certain in vitro systems deficient in ribosome-releasing factor.

Transfer RNA selection at the ribosomal A and P sites

We have examined ribosomal selectivity among tRNAs using an equilibrium competition binding assay. Specifically, we evaluated the dissociation constants of tRNA Phe, tRNA Ile, tRNA fMet, tRNA Val, tRNA Arg, and tRNA Glu, for the A site and, separately, for the P site by measuring competition with [ 3H]phenyl-lactyl-tRNA Phe. We were able to distinguish A and P sites using the site-specific poisons, edeine and tetracycline. In the presence of poly(U), the affinity of tRNA Phe for the P site was significantly greater than that of any of the other five tRNAs. Moreover, the affinities of the tRNAs for the P site varied over three orders of magnitude and paralleled the predicted order of affinities based on potential codon-antidocon interactions. This strongly indicates coding capacity at the P site. In marked contrast, tRNA binding at the A site of poly(U)-programmed ribosomes indicated that tRNA Phe bound only ten- to 20-fold stronger than several non-complementary tRNAs, suggesting that the equilibrium properties of the bare ribosomal A site are very different from the P site. We propose that, in vivo, ribosomal contributions to the accuracy of protein synthesis include both specificity in tRNA selection at the A site and the coding capacity of the P site, as these sites are defined by in vitro criteria. Even at best, however, the precision of the ribosomes of the three strains we have examined is not better than the precision of unaided codon-anticodon interactions. This sets new constraints on the sources of accuracy in protein synthesis. We have also measured the relative affinities of tRNAs for the P site in the absence of message. By comparison of these with the codon-instructed P site, we conclude that message confers an additional affinity for a tRNA possessing a matching anticodon, rather than strongly rejecting tRNAs whose codons do not match. All the data we have are consistent with a large, almost non-specific affinity for tRNAs in both P and A sites, to which a smaller specific contribution is made by codon-anticodon base-pairing.

The evolution of the protein synthesis system.I. A. Model of a primitive protein synthesis system

A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

The evolution of the protein synthesis system. I. A model of a primitive protein synthesis system.

A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

Ribosome slowed by mutation to streptomycin resistance.

ALTHOUGH the basic mechanisms of protein synthesis are now rather well understood, particularly in Escherichia coli, it is less clear how the high accuracy of this process is achieved. (The error rate in protein synthesis in E. coli seems to be about one in 104 amino acid misincorporations (Edelman and Gallant, unpublished results, and our own unpublished data)). A major site determining accuracy in protein synthesis is the ribosome, and in addition to the ribosome-mediated effects on accuracy (in vivo and in vitro) of various metal ions, aminoglycoside antibiotics and organic solvents, there are ribosomal mutations which increase or decrease accuracy2. Certain mutations of the 30S ribosomal protein SI2 conferring streptomycin resistance (the strA locus) enhance accuracy3–5, whereas certain mutations in the S4 protein (the ram locus) are known to decrease accuracy6. The strA mutations are restrictive (reducing) in their effect on both missense and nonsense suppression in vivo and on miscoding in vitro3, the ram mutation, on the other hand, has the opposite effect in both cases6. It is possible that it is the kinetics of polypeptide synthesis at the ribosome that determines accuracy and that the kinetics in turn are affected by the strA and ram mutations. Because the same transfer RNA (tRNA) discrimination kinetics that may determine accuracy may also contribute to the elongation speed, we have investigated the effect of mutation to streptomycin resistance on the speed of polypeptide elongation.

Aging and accuracy of protein synthesis in man: search for inactive enzymatic cross-reacting material in granulocytes of aged people.

Enzyme activities and immunological reactivities of seven different enzymes have been assayed in the polymorphonuclear leukocytes from 10 newborns, 10 aged (over 80 years old) and 10 young adults (20-30 years old). Polymorphonuclears have been chosen because both in the newborns, and in the aged people, they are young cells whose life span is a few hours only. Five cytoplasmic enzymes have been immunologically studied by means of monospecific antienzyme sera: leukocyte pyruvate kinase by immunoinactivation, glucose-6-phosphate dehydrogenase and glucose phosphate-isomerase by electroimmunodiffusion, lactic dehydrogenase (muscle-type) by radial immunodiffusion. Two lysosomal enzymes (alpha-mannosidase and beta-glycuronidase) have been titrated immunologically with the same polyvalent antihuman leukocyte rabbit serum. Small but significant differences in activity were observed for several of the enzymes. By contrast, no significant difference was found, for any of the seven enzymes, as regards the ratio enzyme activity/immunological reactivity, between newborns, young adults and aged people. These results of not support the theory that a decreased accuracy of the protein synthesis machinery is responsible for aging and death. By contrast, the authors emphasize that the enzyme abnormalities reported by various authors in old animals or in old fibroblast cultures closely resemble those demonstrated to be "post-translational modifications" in the case of human glucose-6-phosphate dehydrogenase.

Progressive decrease in protein synthesis accuracy induced by streptomycin in Escherichia coli.

THE passage of genetic information from gene to polypeptide does not proceed with perfect fidelity1–3, but it is not known what short and long-term cellular responses may be induced by decreases in the accuracy of protein synthesis. Errors in gene expression may tend to increase their own rate of production4, and thus could constitute a limiting instability of cellular life. The synthesis of inaccurate RNA, RNA polymerases, tRNAs, tRNA modifying enzymes, tRNA charging enzymes and ribosomal proteins are potential pathways for the perpetuation or amplification of errors. Such errors may be inducible in vivo by external chemical agents and may constitute an indirect pathway for mutagenesis5–8. An enhanced rate of such errors may be the mechanism by which certain deleterious mutations exert their effects. Here we report the results of experiments with Escherichia coli designed to detect evidence of self amplification of errors in protein synthesis. If such error feedback is quantitatively significant, a slight increase in the pre-existing error rate should be followed by a progressive decrease in the accuracy of protein synthesis. While this process could lead to a new stable level and thus need not precipitate a catastrophic breakdown, some late effects should be observable if the mechanisms are at all as hypothesised.

Catalytic role for RNA in DNA replication

THERE are two biochemical systems which probably evolved before the development of accurate polynucleotide-specified protein synthesis: these are the system for polynucleotide replication and the machinery of protein synthesis itself1, 2. Before accurately specified proteins became available, these processes were perhaps catalysed by polynucleotide enzymes. Both tRNA and rRNA, which can be viewed as polynucleotide enzymes, have persisted as indispensable components of the contemporary apparatus. This has led me to wonder whether polynucleotide enzymes might still be operative in DNA replication. Moreover, in view of the complexity which would have been required for even a rudimentary form of protein synthesis, it seems unlikely that tRNA and rRNA arose by chance in a single evolutionary step1. More probably they have evolved from the replicative machinery for polynucleotides and thus it seems likely that the machinery of DNA replication may have many features in common with the polynucleotide components of protein synthesis.

Senescence and the Fidelity of Protein Synthesis in Drosophila

The possibility that ageing may be a consequence of the failure of cells to maintain accurate protein synthesis has prompted experiments in which the larvae of Drosophila melanogaster are treated with agents expected to induce errors in protein synthesis. Treatment of larvae with amino-acid analogues or with streptomycin does indeed decrease the life-span of adults.