Mistranslation in twelve Escherichia coli ribosomal proteins. Cysteine misincorporation at neutral amino acid residues other than tryptophan.

The misincorporation of cysteine (codon: UGU/C) into twelve ribosomal proteins devoid of cysteine has been studied. Although it is generally assumed that cysteine is misincorporated at arginine and tryptophan residues (codons: CGU/U and UGG respectively), our results are consistent with the idea that cysteine is also misincorporated at phenylalanine residues (codon: UUU/C) through a second-position C:U mismatch. Cysteine was found in ribosomal proteins L29, L32/L33 and S10, under conditions where only its misincorporation at neutral residues was measured. Since these proteins contain no tryptophan, the date imply that cysteine has replaced a neutral amino acid other than tryptophan. Because there was a statistically significant correlation between the total level of cysteine in the twelve proteins under study and their content of phenylalanine and arginine residues, we conclude that there is a likelihood of cysteine misincorporation at phenylalanine residues, in addition to its misincorporation at arginine and tryptophan residues. Our measurements are consistent with the existence of a cluster of ribosomal proteins having an average mistranslation frequency of 2.5 X 10(-4)/residue and another having an average mistranslation frequency of 10(-3)/residue. There was three times less cysteine misincorporated into ribosomal protein L1 than into L7/L12, although the L1 mRNA contains eleven CGU/C codons and four UUU/C codons while the L7/L12 mRNA contains only one arginine and two phenylalanine codons (both proteins are free of tryptophan). Furthermore, the mRNAs for both L1 and L7/L12 contain a CGU codon located in the context GUA-codon-GG and there was as much cysteine incorporated at this codon in L7/L12 [Bouadloun, F., Donner, D. and Kurland, C.G. (1983) EMBO J. 2, 1351-1356] than in the whole of L1. This suggests that, relatively speaking, little cysteine is to be found at the phenylalanine and the other ten arginine positions of L1 and that the phenylalanine residues of L7/L12 are particularly error-prone.

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: under in vitro conditions in which translation fidelity was similar to what is known about the accuracy of translation in vivo; and under stresses of varying Mg2+ concentrations (3-12 mM), pH (6.6-8.4), temperature (26-42 degrees 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: in liver and hippocampus extracts from 7- and 33-month-old rats; and in brain extracts from 3- and 29-month-old rats. The results 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 Mg2+ concentrations (3 to 22 mM), various pH's (6.6 to 8.6), various temperatures (23 to 42 degrees 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.

Effect of ethanol, phenol, formamide, dimethyl sulfoxide, paromomycin, and deuterium oxide on the fidelity of translation in a brain cell-free system.

The effects of six different agents (ethanol, phenol, formamide, dimethyl sulfoxide, heavy water, and a misreading-inducing antibiotic, paromomycin) on the activity and the accuracy of poly(U) translation have been compared under a range (2.5-12 mM) of Mg2+ concentrations in a rat brain cell-free system. The effect of most of these agents was remarkably sensitive to the Mg2+ concentration under which the assay was made. Ethanol decreased the fidelity of translation, and the efficiency of ethanol was increased 3-10-fold by higher Mg2+ concentrations. The effect of paromomycin was identical with that of ethanol, despite its very different structure. Formamide, a "RNA denaturant", increased the accuracy of translation under all Mg2+ concentrations tested. Dimethyl sulfoxide, another type of RNA denaturant, decreased the accuracy of translation under all Mg2+ concentrations tested. Phenol increased the accuracy of translation at high Mg2+ concentrations but decreased it at low Mg2+ concentrations. D2O did not change to any appreciable extent the accuracy of translation, at all the Mg2+ concentrations used. There exists a cooperativity between the effects of Mg2+ and ethanol, Mg2+ and paromomycin, and Mg2+ and dimethyl sulfoxide on the fidelity of translation; no such cooperativity was detected between Mg2+ and formamide and between Mg2+ and D2O. The differential effects of dimethyl sulfoxide and formamide are interpreted in terms of their different dielectric constants. The dielectric constant of dimethyl sulfoxide is higher than that of water, while that of formamide is low er.

The activity of cholesterol ester hydrolase in the enzymatic determination of cholesterol: comparison of five enzymes obtained commercially.

The use of five cholesterol ester hydrolases (CEH), numbered 1 to 5, for the enzymatic determination of total cholesterol of human and rat serum are compared. All CEH gave approximately the same value (no statistical difference) for human serum. However, when rat serum cholesterol was determined, CEH-2 yielded a value significantly lower when compared to the four other CEH. The ability of each CEH to hydrolyze individual cholesterol esters was tested. During a 15-min incubation, all CEH were capable of hydrolyzing nearly 100% of cholesteryl oleate and linoleate. In contrast, the hydrolysis of cholesteryl arachidonate was only partial and varied from 20 to 80% depending on the CEH used. The highest hydrolysis was obtained by CEH-1 while the value given by CEH-2 was only 22% of that obtained by CEH-1. The rate of hydrolysis of cholesteryl arachidonate differed markedly among the CEH. The CEH-2-hydrolyzed the cholesteryl arachidonate at a rate seven times lower than the rate obtained with CEH-1. The data suggest that, Under our incubation conditions, CEH-2 did not properly hydrolyze the cholesteryl arachidonate. This phenomenon may be crucial whenever total cholesterol has to be determined enzymatically in the serum of species that contain large amount of cholesteryl arachidonate such as rat, mouse, or dog serum.