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1.
Emmanuelle Boy Françoise Borne Jean-Claude Patte 《Molecular & general genetics : MGG》1978,159(1):33-38
Summary When studying mutants affecting lysyl-tRNA synthetase or tRNALys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This excludes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNALys is a secondary effector involved in the regulation of the synthesis of this enzyme.Abbreviations DAP
diaminopimelate
- KRS
lysyl-tRNA synthetase
- L-lysine
tRNA ligase (AMP) (EC6.1.16)
- AK III
lysinesensitive aspartokinase (EC 2.7.24)
- ASA-dehydrogenase
aspartic semialdehyde dehydrogenase (EC 1.2.1.10)
- DHDP-reductase
dihydrodipicolinic acid reductase
- DAP-decarboxylase
diaminopimelate decarboxylase (EC 4.1.1.20)
- AK I
threonine-sensitive aspartokinase
- HDHI
threonine-sensitive homoserine dehydrogenase 相似文献
2.
The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain. 相似文献
3.
M Di Girolamo V Busiello A Di Girolamo C Foppoli C De Marco 《Biochemistry international》1988,17(3):545-554
A thialysine-resistant mutant of the E. coli KL16 strain was isolated. It can grow equally well in the presence and in the absence of thialysine. The properties of the two lysine transport systems, of the lysyl-tRNA synthetase and of the aspartokinase III (AK III) were studied in the mutant and in the parent strain. AK III is the first enzyme of the lysine biosynthetic pathway and its activity is involved in the regulation of lysine biosynthesis by feed-back and repression mechanism. No difference between the two strains was evidenced as regards 1) the affinity of the transport systems for lysine and thialysine 2) the activity of the lysyl-tRNA synthetase 3) the allosteric inhibition of the AK III by lysine and thialysine. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the enzyme is much less repressed both by lysine and thialysine. The possible correlation between the activity of AK III and the thialysine-resistance is discussed in this paper. 相似文献
4.
V Busiello C Cini R Coccia A Di Girolamo M Di Girolamo 《Physiological chemistry and physics and medical NMR》1988,20(3):193-197
A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain. 相似文献
5.
Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity. 相似文献
6.
Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro 总被引:6,自引:0,他引:6
F Lustig P Elias T Axberg T Samuelsson I Tittawella U Lagerkvist 《The Journal of biological chemistry》1981,256(6):2635-2643
The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides. 相似文献
7.
Fern T. Coles Jean E. Brenchley 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,428(3):647-655
Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition caused by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzymes synthesis as well as inhibition of activity. 相似文献
8.
Sandro F Ataide Sharnise N Wilson Sandy Dang Theresa E Rogers Bappaditya Roy Rajat Banerjee Tina M Henkin Michael Ibba 《ACS chemical biology》2007,2(12):819-827
Structural and functional diversity among the aminoacyl-tRNA synthetases prevent infiltration of the genetic code by noncognate amino acids. To explore whether these same features distinguish the synthetases as potential sources of resistance against antibiotic amino acid analogues, we investigated bacterial growth inhibition by S-(2-aminoethyl)-L-cysteine (AEC). Wild-type lysyl-tRNA synthetase (LysRS) and a series of active site variants were screened for their ability to restore growth of an Escherichia coli LysRS null strain at increasing concentrations of AEC. While wild-type E. coli growth is completely inhibited at 5 microM AEC, two LysRS variants, Y280F and F426W, provided substantial resistance and allowed E. coli to grow in the presence of up to 1 mM AEC. Elevated resistance did not reflect changes in the kinetics of amino acid activation or tRNA (Lys) aminoacylation, which showed at best 4-6-fold improvements, but instead correlated with the binding affinity for AEC, which was decreased approximately 50-fold in the LysRS variants. In addition to changes in LysRS, AEC resistance has also been attributed to mutations in the L box riboswitch, which regulates expression of the lysC gene, encoding aspartokinase. The Y280F and F426W LysRS mutants contained wild-type L box riboswitches that responded normally to AEC in vitro, indicating that LysRS is the primary cellular target of this antibiotic. These findings suggest that the AEC resistance conferred by L box mutations is an indirect effect resulting from derepression of lysC expression and increased cellular pools of lysine, which results in more effective competition with AEC for binding to LysRS. 相似文献
9.
Roles of the two lysyl-tRNA synthetases of Escherichia coli: analysis of nucleotide sequences and mutant behavior. 总被引:15,自引:11,他引:4
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The complete nucleotide sequence of lysU, the gene for the heat-inducible lysyl-tRNA synthetase of Escherichia coli, was determined and compared with the published sequence of lysS (herC), the gene for the constitutive lysyl-tRNA synthetase. These unlinked genes were found to be identical over 72% of their lengths. The deduced amino acid sequences of the respective gene products, LysU and LysS, were identical over 85% and similar over 92% of their lengths. Accumulation of high levels of LysU during growth of strains carrying the wild-type allele of lysU on multicopy plasmids had no observable effect on growth or on the synthesis of LysS. A lysU deletion strain was constructed and was shown to grow normally at low temperature (28 degrees C) but poorly at 44 degrees C; the slow growth (45% of normal) at elevated temperature was fully reversed by plasmids bearing wild-type lysU. The implications of these findings for the existence of two aminoacyl-tRNA synthetases for lysine are discussed. 相似文献
10.
The affinity column lysyldiaminohexyl-Sepharose 4B has been synthesized for the purification of aminoacyl-tRNA synthetase complexes. Lysyl-tRNA synthetase (EC 6.1.1.6) bound specifically to the Sepharose-bound lysine. The purified lysyl-tRNA synthetase was associated with arginyl-tRNA synthetase (EC 6.1.1.16) and sedimented at 18S and 12S. A 24S lysyl-tRNA synthetase bound specifically to the affinity column and also found associated with arginyl-tRNA synthetase. The results favor the model of a heterotypic multienzyme complex of mammalian aminoacyl-tRNA synthetases. 相似文献
11.
The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain. 相似文献
12.
Henry S. Simms 《The Journal of general physiology》1928,12(2):231-239
The titration data of edestin show that all the arginine found on hydrolysis exists in this protein as "prearginine." The extra ionizable groups of histidine, lysine and tyrosine are free in the quantities found on hydrolysis. Part of the extra carboxyl groups of aspartic and glutamic acids are bound as amides, and 50 per cent are bound in some other manner (perhaps anhydride) leaving only about 6 per cent of these groups free to ionize in edestin. The prearginine in edestin is not converted into arginine on hydrolysis with pepsin up to 18 per cent (of the total hydrolysis). In more highly hydrolyzed solutions it is not possible to detect such a conversion, due to high buffering. Complete hydrolysis however converts prearginine into arginine which can be isolated. Hydrolyzed edestin promotes the growth of sarcomatous fibroblasts about equally well whether 5, 14 or 18 per cent hydrolyzed. 相似文献
13.
Brevet A Chen J Commans S Lazennec C Blanquet S Plateau P 《The Journal of biological chemistry》2003,278(33):30927-30935
The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases. 相似文献
14.
Gene for Heat-Inducible Lysyl-tRNA Synthetase (lysU) Maps near cadA in Escherichia coli 总被引:28,自引:16,他引:12
Ruth A. VanBogelen Vicki Vaughn Frederick C. Neidhardt 《Journal of bacteriology》1983,153(2):1066-1068
A hybrid ColE1 plasmid from the Clarke-Carbon colony bank with a 7-kilobase insertion was found to encode the inducible lysyl-tRNA synthetase along with the catabolic enzyme lysine decarboxylase. The gene for the inducible synthetase, lysU, must lie within 0.3 min of the lysine decarboxylase gene, cadA, at 92 min on the Escherichia coli genetic map. 相似文献
15.
Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition casued by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzyme synthesis as well as inhibition of activity. 相似文献
16.
Background
Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. 相似文献17.
A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate. 相似文献
18.
Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography 总被引:3,自引:2,他引:1
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Barbara S. Vold 《Journal of bacteriology》1970,102(3):711-715
Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons. 相似文献
19.
20.
M Hassani D H Pincus G N Bennett I N Hirshfield 《Applied and environmental microbiology》1992,58(8):2704-2707
The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C. This induction does not appear to be under HtpR control. 相似文献