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1.
There are two possible mechanisms (co- or post-translational) for incorporation of Se into glutathione peroxidase in which selenocysteine presents at the active site of the enzyme and corresponds to UGA on the mRNA. We studied the above mechanisms using opal suppressor tRNA in mammals. Opal suppressor tRNA did not accept any selenocysteine and phosphoseryl-tRNA did not change to selenocysteyl-tRNA. Meanwhile, phosphoprotein changed to a protein containing selenocysteine by the incubation with H2Se and some enzymes. From these results, we propose that phosphoserine on glutathione peroxidase (apo-enzyme), which is synthesized with phosphoseryl-tRNA, is converted to selenocysteine in the mature enzyme, by a posttranslational mechanism. Opal suppressor tRNA may play a role to synthesize the apo-enzyme of glutathione peroxidase. 相似文献
2.
Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism 总被引:9,自引:0,他引:9
The opal termination codon UGA is used in both prokaryotic and eukaryotic species to direct the specific insertion of selenocysteine into certain selenium-dependent enzymes. So far a formate dehydrogenase (hydrogenase-linked) of Escherichia coli and glutathione peroxidases of murine, human and rat origin have been identified as enzymes containing selenocysteine residues encoded by UGA. A novel seryl-tRNA, anticodon UCA, that specifically recognizes the UGA codon is required for selenocysteine incorporation into formate dehydrogenase. A eukaryotic UGA suppressor tRNA with UCA anticodon that accepts serine and is phosphorylated to O-phosphoseryl-tRNA may have a corresponding function in glutathione peroxidase synthesis. Other factors required for the unusual usage of the in-frame UGA codons to specify selenocysteine incorporation and the biochemical mechanism involved in distinguishing these from normal UGA termination codons are discussed. 相似文献
3.
Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively 总被引:4,自引:0,他引:4
An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes. 相似文献
4.
Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA 总被引:26,自引:0,他引:26
B J Lee P J Worland J N Davis T C Stadtman D L Hatfield 《The Journal of biological chemistry》1989,264(17):9724-9727
The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%). 相似文献
5.
Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe 总被引:3,自引:0,他引:3
The mechanisms of the synthesis of mammalian selenocysteyl-(Scy)-tRNA were studied using [75SE]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy-tRNA. [75Se]Scy, liberated from aminoacyl-tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy-tRNA synthesis was found in the supernatant at 105,000 x g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl-tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNA(IGA) is not a substrate on which to synthesize Scy-tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy-tRNA preparation on Sephacryl S-200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE-cellulose did not contain tRNA kinase, therefore Scy-tRNA must be directly synthesized from seryl-tRNA, not through phosphoseryl-tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324]. 相似文献
6.
Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA). 相似文献
7.
The selC gene product, tRNA(Sec), inserts selenocysteine at UGA (opal) codons in a specialized mRNA context. We have investigated the action of the tRNA at ordinary UGA codons, normally not translated, by changing the unusual structural features of tRNA(Sec). Sequences in the D arm, CCA arm and variable arm of the tRNA all contribute to the prohibition against translation of ordinary UGA codons. One multiple mutant is a moderately efficient serine-inserting UGA suppressor tRNA. 相似文献
8.
Possible incorporation of phosphoserine into globin readthrough protein via bovine opal suppressor phosphoseryl-tRNA 总被引:5,自引:0,他引:5
Suppressor [32P]phosphoseryl-tRNA, prepared using bovine seryl-tRNA synthetase and ATP:seryl-tRNA phosphotransferase, was mixed with rabbit reticulocyte lysates containing endogenous hemoglobin mRNA having the termination codon UGA (opal). The chromatographic pattern of the lysate on Sephacryl S-200 showed that the radioactivity of [32P]phosphate in the hot trichloroacetic acid-precipitate (phosphoprotein) was eluted at the position between mature hemoglobin and globin subunits. The phosphoprotein, obtained by chromatography on S-200, moved to the position corresponding to that of globin readthrough protein on SDS-PAGE. The analyses of the hydrolyzate of the phosphoprotein showed the presence of phosphoserine in the protein. These results suggest that animal opal suppressor tRNA functions in vitro to transfer phosphoserine to the position of the termination codon UGA (opal) on mRNA. 相似文献
9.
Specific occurrence of selenium in enzymes and amino acid tRNAs 总被引:9,自引:0,他引:9
T C Stadtman 《FASEB journal》1987,1(5):375-379
In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium. 相似文献
10.
Role of acidic residues as substrate determinants for casein kinase I 总被引:17,自引:0,他引:17
Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase. 相似文献
11.
In vitro synthesis of glutathione peroxidase from selenite. Translational incorporation of selenocysteine 总被引:3,自引:0,他引:3
The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase. 相似文献
12.
Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom. 总被引:16,自引:2,他引:14
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B J Lee M Rajagopalan Y S Kim K H You K B Jacobson D Hatfield 《Molecular and cellular biology》1990,10(5):1940-1949
Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought. 相似文献
13.
Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product 总被引:1,自引:0,他引:1
Giafen T. Chen Milton J. Axley Joseph Hacia Masayori Inouye 《Molecular microbiology》1992,6(6):781-785
The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H. 相似文献
14.
Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene. 总被引:25,自引:4,他引:21
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Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes. 相似文献
15.
Wayne C. Hawkes Al L. Tappel 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,739(2):225-234
The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase 相似文献
16.
用诱变剂苯甲基磺酰氟(PMSF)活化兔抗人IgM(Fcμ)片段上特殊活性部位的丝氨酸(Ser)残基,经硒化氢(H_2Se)处理,则将丝氨酸转变成硒代半胱氨酸(SeCys)。诱变后的抗体具有谷胱甘肽过氧化物酶(GSH-Px)活性,其活性为世界最好的GSH-Px模拟物PZ51的70多倍。抗体效价为1:8,与没诱变的抗体相似。 相似文献
17.
Wayne C. Hawkes David E. Lyons Al L. Tappel 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(3):183-191
The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteme, the most highly purified 75Se-fractions were > 100-fold purified relative to 35S. These fractions contained < 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system. 相似文献
18.
Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells
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We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells. 相似文献
19.
Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium. 总被引:11,自引:1,他引:10
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Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions. 相似文献
20.
Phosphorylation of threonine and serine residues of native and partially dephosphorylated caseins by a rat liver cyclic AMP-insensitive protein kinase.
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The preferential phosphorylation of threonine residues of native casein fractions by a rat liver cyclic AMP-independent protein kinase (EC 2.7.1.37) is abolished by preliminary limited dephosphorylation of the substrates, which promotes a fall in the phosphothreonine/phosphoserine ratios from values higher than 1 to much less than 0.1. This finding and the identification of the threonine residues phosphorylated support the view that the liver protein kinase affects threonine residues only when suitable serine residues, which fulfil the structural requirements for attack by the enzyme but which are not yet phosphorylated, are not available. 相似文献