共查询到20条相似文献,搜索用时 62 毫秒
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Roland Klassen Pia Grunewald Kathrin L. Thüring Christian Eichler Mark Helm Raffael Schaffrath 《PloS one》2015,10(3)
In eukaryotes, wobble uridines in the anticodons of tRNALys
UUU, tRNAGlu
UUC and tRNAGln
UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALys
UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALys
UUU hypomodification and malfunction. 相似文献
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Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)5s2U34 in mitochondrial tRNALys, tRNAGlu, and tRNAGln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm5 group (cmnm5U34), while Mto2p is responsible for the 2-thiouridylation (s2U34) of these tRNAs. Previous studies showed that partial modifications at U34 in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNALys, tRNAGlu, and tRNAGln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U34 of tRNALys, tRNAGlu, and tRNAGln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm5U34 by the mto1 or mss1 mutation. The complete loss of modifications at U34 of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U34 of tRNALys, tRNAGlu, and tRNAGln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function. 相似文献
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Dietmar Porschke 《Biophysical chemistry》1979,10(1):1-16
The interactions between basic oligopeptides (Lys2, Lys3, Arg2, and Arg3) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescencs followed by coacervation- Arg3 causes coacervation in all polynucleotides used, yet Lys3 only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg3 (150 μM) than for Lys3 (500 μM). 2) Medium concentrations (?10 μM) of Arg3 and Lys3 induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys3, but not by Arg3. 3) The onset of peptide association is observed at very low peptide concentrations (?1 μM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants kR and kD. Using a simple model with independent and “separate” binding sites, K for Arg3 and Lys3 is found to be in the range of 106 to 107 M?1. In the case of Arg2 and K is lower by a factor of about 10. For various polynucleotides KArg3 is slightly higher than KLys3. except in the case of poly(I), where KArg3/KLys3 ≈ 5. Similar data are obtained by application of a “sphere model” (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg3: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interaction Both field jump and stopped flow data demonstrate a high mobility of the peptide lisands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a “sphere” model, which considers a) excluded binding under the condition of fast Ugand distribution along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 × 1011 M?1 s?1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers. 相似文献
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Di You Li-li Yao Dan Huang Jorge C. Escalante-Semerena Bang-Ce Ye 《Journal of bacteriology》2014,196(17):3169-3178
Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD+-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys237, Lys380, Lys611, and Lys628 was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. 相似文献
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Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNALys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNALys did not alter the percentages of the free-, charged- or peptidyl-tRNALys species compared with those found in strains that did not overproduce tRNALys. At 41°C, however, despite increases in the level of peptidyl-tRNALys, the excess tRNALys helped to maintain the concentration of charged-tRNALys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNALys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNALys and the causes of tRNALys starvation in pth(Ts) strains grown at non-permissive temperatures are considered. 相似文献
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A series of sequential polypeptides, (Lysi-Alaj)n, and random copolypeptides, (Lysx, Alay)n, were synthesized. The competitive effect of Ala, a helix former, and Lys, whose homopolymer has a β-form in neutral NaDodSO4 solution, was determined by CD and absorption spectroscopy. All the polypeptides studied were unordered in neutral solution without the surfactant. Of the six sequential polypeptides only (Lys-Ala)n adopted a stable β-form in NaDodSO4 solution. Most striking is the difference between this polypeptide, (Lys2-Ala2)n and (Lysx, Alay)n, even though they all have equimolar Lys and Ala. (Lys2-Ala2)n was partially helical in 2.5–5 mM NaDodSO4 but approached the unordered form in 50 mM NaDodSO4, whereas (Lys50, Ala50)n was completely helical in all NaDodSO4 concentrations. Even Lysrich (Lys2-Ala)n and (Lys3-Ala)n formed a partial helix and a trace of the β-form, respectively, in low NaDodSO4 concentrations; both reverted to the unordered form in high NaDodSO4 concentrations. These results can be explained by Pauling-Corey's model for β-pleated sheets. Only (Lys-Ala)n has all DodSO-bound Lys+ residues on one side and Ala residues on the other side of the polypeptide chain. They can nestle quiet efficiently in a β-sheet and between neighboring β-sheets. Our results further imply that random copolypeptides are not completely random; they comprise varying segments of (Lysk-Alam), where k and m could vary from zero to a small integer. 相似文献
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Kirk Hayenga Charles Hedgcoth Mary Harrison Victor K. Lin B. J. Ortwerth 《Molecular and cellular biochemistry》1986,71(1):25-30
Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA2
Lys and tRNA4
Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA4
Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNA4ys is not a simple precursor of tRNA2
Lys. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA2
Lys and tRNA4
Lys were obtained. The distinctions between tRNA2
Lys and tRNA4
Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P1,P4-tetraphosphate, a putative signal in DNA replication. 相似文献
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The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg334, Arg396 and Arg638 were directly assigned to the ETF and ii) Arg198 was assigned as the only tryptic site to the LTF. Arg671, Lys712/Lys713 and Lys728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl2, EGTA or AlF4− strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg1002 as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes. 相似文献
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Larry C. Waters James R. Burke M.P. Stulberg 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(2):201-207
Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNAArg, 51°C, 1.6; tRNALys, 57°C, 0.7 and tRNATrp, 76°C, 1.0. Possible functions for the non-primer tRNAs (tRNAArg and tRNALys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a tm of 56°C and is consistent with the hypothesis that tRNALys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNAArg or tRNALys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNAArg and tRNALys. 相似文献
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A ferredoxin of MW 11 000 was isolated from the marine alga Rhodymenia palmata (Palmaria palmata). In its oxidised form the ferredoxin had absorption maxima at 276, sh 281, 328, 423 and 465 nm, and contained a single [2Fe-2S] cluster. The midpoint potential of the ferredoxin was ?400 mV and it effectively mediated electron transport in NADP+-photoreduction by higher plant chloroplasts, and pyruvate decarboxylation by the phosphoroclastic system of an anacrobic bacterium. The amino acid composition was Lys3, His1, Arg1, Asx12, Thr9, Ser8, Glx13, Pro4, Gly8, Ala7, Cys5, Val8, Ile4, Leu9, Tyr4, Phe2; tryptophan and methionine were absent from the molecule. The N-terminal amino acid region consisting of ca half the total amino acid sequence was determined using an automatic sequencer. 相似文献
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A series of sequential polypeptides (LysiRj)n (R is Leu, Ser, or Gly) and random copolypeptides, (Lysx, Leuy)n, were synthesized. Their conformation in NaDodSO4 solution was determined by CD. Only (Lys-Leu)n, (Lys-Ser)n, and (Lys3-Ser)n adopt a stable β-form in the surfactant solution; (Lys-Ser2)n, (Lys-Ser3)n, (Lys2-Ser2)n, and (Lys2-Ser)n have an unstable β-form, which reverts to an unordered form in high NaDodSO4 concentrations, even though both Ser and DodSO-bound Lys+ are β-formers. In contrast, (Lys-Gly)n remains unordered in NaDodSO4 solution. On the other hand, Lys-rich (Lys2-Leu)n forms an unstable helix and (Lys2-Leu2)n a stable helix in NaDodSO4 solution. In 25 mM NaDodSO4 (Lysx, Leuy)n also forms a helix up to x = 75 and reverts to the β-form at x = 90. This compares with the helical conformation of (Lysx, Alay)n up to x = 65 and its β-form at x = 90, suggesting that Leu is an even stronger helix-former than Ala. Our results may provide a plausible explanation for the increase in helicity and disruption of the β-form for many proteins in NaDodSO4 solution, that is, the polypeptide chain of a protein usually favors a helical conformation over a β-form in the presence of excess surfactant. 相似文献
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To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg243, Lys248, Tyr264, or simultaneously at Lys6 and Lys19, or Lys6 and Lys23 caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys6 and Lys23 changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg243 changed to Ala) and RecA-Y264A (Tyr264 changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg243 is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr103 and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure. 相似文献
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Joost Schimmel Crina I.A. Balog André M. Deelder Jan W. Drijfhout Paul J. Hensbergen Alfred C.O. Vertegaal 《Journal of Proteomics》2010,73(8):1523-1534
Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the ε-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110 kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys94 and Lys141 of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys94 and Lys141, but not Lys709 and Lys742, resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys94. These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites. 相似文献
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A 22-residue peptide toxin from the venomous marine snail Conus geographus (L.) was found to have a most unusual amino acid composition: Lys4, Arg3, , Asx2, Glx2, Thr, Ala, plus three residues of trans-4-hydroxyproline. Absence of Gly and Pro indicates that the hydroxyproline must be in sequences different from those in which hydroxyproline occurs in collagen and other proteins. 相似文献