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) X 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 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 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.     

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.     

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent (relA+)/relaxed (relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.     

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.     

Bacillus subtilis mutants dependent on streptomycin

Summary Six streptomycin-dependent mutants of Bacillus subtilis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.     

The increase by spermidine of fidelity of protamine synthesis in a wheat-germ cell-free system

The influence of spermidine on the fidelity of natural mRNA-directed protein synthesis has been investigated. With protamine mRNA as a template for protamine synthesis, misincorporation of lysine, histidine, threonine and cysteine for arginine was measured in the presence and absence of spermidine. It was found that misincorporation of these four amino acids in the presence of spermidine was less than or nearly equal to that occurring in the absence of spermidine; however, incorporation of arginine was stimulated greatly by spermidine. These results clearly show that spermidine induced an increase of fidelity in protamine synthesis. The increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes. The frequency of misreading the 5' base of the codon (misincorporation of cysteine) was greater than that of the middle base of the codon (misincorporation of histidine), but spermidine reduction of misreading was more marked at the middle base of the codon. Misincorporation of lysine (misreading of G to A residue at the middle base of the codon) was greater than that of threonine (misreading of G to C residue), but spermidine reduction of misreading was more marked in the misincorporation of threonine. It was deduced from these results that spermidine inhibited low-frequency misreading more effectively than high-frequency misreading.     

The [(99m)Tc(N)(PNP)](2+) metal fragment: a technetium-nitrido synthon for use with biologically active molecules. The N-(2-methoxyphenyl)piperazyl-cysteine analogues as examples

The incorporation of a bioactive molecule into a nitrido-containing (99m)Tc-complex has been successfully achieved by using the [TcN(PNP)](2+) metal fragment. In this strategy, the strong electrophilic [TcN(PNP)](2+) metal fragment efficiently reacts with bifunctional chelating ligands having a pi-donor atom set, such as N-functionalized O,S-cysteine. The 2-methoxyphenylpiperazine (2-MPP) pharmacophore, which displays preferential affinity for 5HT(1A) receptors, was conjugated to the amino group of cysteine to obtain 2-MPPP-cys-OS, where 2-MPPP is 3-[4-(2-methoxyphenyl)piperazin-1-yl]propionate. The asymmetric Tc(V)-nitrido complexes, [(99g/99m)Tc(N)(PNP)(2-MPPP-cys-OS)] (PNP = PNP3, PNP4), were obtained in high yield (95%), by simultaneous addition of PNP and 2-MPPP-cys-OS ligand to a solution containing a starting (99g)/(99m)Tc-nitrido precursor. A mixture of syn and anti isomers was observed, the latter being the thermodynamically favored species. In vitro challenge experiments using the anti isomers with glutathione and cysteine indicated that no transchelation reaction occurs. Assessment of the in vitro 5HT(1A) receptor-affinity of the technetium complexes revealed that only the anti-PNP4 complex possesses some affinity for the receptor, but displayed negligible brain uptake in biodistribution studies in rats in vivo.     

Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity

Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa. The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [(3)H]lysine (15% of maximum activity) and [(3)H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.     

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.     

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.     

Chemical modifications of the sigma subunit of the E. coli RNA polymerase.

The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.     

Fluorescence studies on a streptomycin-induced conformational change in ribosomes which correlates with misreading

The fluorescent reagent N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) was employed to detect and study the previously reported conformational change in the Escherichia coli ribosome induced by streptomycin. Labeling of ribosomes with this probe, which results in the derivatization of proteins S18 and L31', described earlier, inhibits neither their ribosomal protein synthesizing nor misreading ability. To calculate the amount of streptomycin bound to the ribosome, we determined the K'D for streptomycin, which is 0.24 micron, indicating that under our conditions, bound streptomycin/ribosome molar ratios are low, not in excess of 1. Under these conditions, streptomycin addition induces fluorescence quenching by 15% but does not affect streptomycin-resistant ribosomes. Maximal misreading occurs at these same ratios. Removal of AEDANS-L31' from the ribosomes drastically reduces streptomycin-induced quenching indicating the involvement of the environment of this protein in streptomycin action. The finding that streptomycin decreases AEDANS-L31' affinity for the ribosome supports this view. Streptomycin has been shown to bind to the 30 S subunit protein S4 while the 50 S protein L31' has been shown to be localized at the subunit interface. Thus, the observation that streptomycin influences this 50 S subunit protein L31', combined with the tight correlation between the effects of streptomycin on quenching and on misreading, strongly suggests that this antibiotic induces a conformational change at the subunit interface of the ribosome, and that this results in misreading. Polyuridylic acid also induces a conformational change in the ribosome but the polynucleotide and streptomycin seem to act independently. Streptomycin-resistant ribosomes, which undergo neither streptomycin-induced fluorescence nor streptomycin-induced misreading, are resistant to misreading induced by high Mg2+ as well.     

Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine

We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

Control of basal-level codon misreading in Escherichia coli

Basal-level misreading of asparagine codons was examined in a number of Escherichia coli strains. Lysine substitutions were measured by quantitating the amount of charge heterogeneity in MS2 coat protein. In most strains the heterogeneity was consistent with misreading of AAU codons at a frequency of 3-6 X 10(-3). Strains with streptomycin resistance mutations (rpsL) have reduced levels of misreading. There is no significant difference in the frequency of basal-level errors in stringent (relA+) and relaxed (relA) strains, even during starvation for amino acids unrelated to the substitution being studied.     

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.     

Studies on the Biosynthesis of Streptomycin

Myo-inositol, especially in combination with arginine, enhances streptomycin production. Compounds which show structural relationship with myo-inositol are ineffective.

Myo-inositol decreases the incorporation of C¹⁴-glucose into streptomycin, particularly into streptidine. This effect suggests that myo-inositol is a precursor of the streptidine ring.

Methionine stimulates antibiotic production in a synthetic medium but proves to be unfavorable in a complex medium.

The γ- and δ-isomers of hexachlorocyclohexane inhibit streptomycin formation.

The formation of streptomycin by washed mycelium was studied. Essentially the same results were here obtained as with growing cultures.