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1.
The two major lysine tRNAs from rat liver, tRNA2Lys and tRNA5Lys, were sequenced by rapid gel or chromatogram readout methods. The major tRNA2Lys differs from a minor form only by a base pair in positions 29 and 41; both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Although highly related, the major tRNA2Lys and tRNA5Lys differ in four base pairs and four unpaired nucleotides, including the first position of the anticodons, but have the same base pair in positions 29 and 41. The three tRNAs maintain a m2G-U pair in the acceptor stem. Detection of this m2G is in contrast to other reports of lysine tRNAs. Sequences of lysine tRNAs are strongly conserved in higher eukaryotes.  相似文献   

2.
Ribothymidine, generally considered a universal nucleotide in tRNA, is completely absent in five specific wheat embryo tRNAs. These consist of two species of glycine tRNA and three species of threonine tRNA. These tRNAs, all extensively purified, are acceptable substrates for E. coli - ribothymidine forming-uracil methylase, which produces one mole of ribothymidine per mole of tRNA. These five tRNAs account for about 90% of the wheat embryo tRNAs which are substrates for this methylase. Nucleotide sequence analysis of one of these tRNAs, tRNAGlyI, confirmed both the complete absence of ribothymidine at position 23 from the 3′end, and the presence of uridine at that site instead. In addition, it is shown that methylation with E. coli uracil methylase quantitatively converts uridine at position 23 to ribothymidine, while no other uridine in the molecule is affected.Using E. coli uracil methylase as an assay we have detected this class of ribothymidine lacking tRNA, in each case consisting of a few specific species, in other higher organisms, such as wheat seedling, fetal calf liver and beef liver, in addition to wheat embryo. We could not detect this class of tRNA in E. coli or yeast tRNA.  相似文献   

3.
The 7-methylguanosine (m7G) content of two individual mitochondrial tRNAs, labelled in the aminoacyl moiety was assayed by the specific cleavage of the tRNA at this nucleotide followed by electrophoretic analysis to identify the 3'-terminal fragment of the tRNA. Neither Syriam hamster mitochondrial tRNALeu nor tRNAMet were found to contain m7G. In contrast, cytosolic tRNAMetS were cleaved indicating the presence of m7G, apparently 27--28 and 29 nucleotides from their 3' terminus. Cystolic tRNALeu was not cleaved. These results are discussed in relationship to the reported low content of methylated nucleosides in mitochondrial 4 S RNA.  相似文献   

4.
Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.  相似文献   

5.
Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.  相似文献   

6.
The transfer ribonucleic acids (tRNAs) of B. subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine. About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores. In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis. Thus, both in vitro and in vivo methylation indicates that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification is noted in tRNAs from stationary cells or spores. Hence, tRNA modifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.  相似文献   

7.
Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m1G9 and m1A9 formation. The ability of the mitochondrial tRNA:m1R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.  相似文献   

8.
tRNA(guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles, Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D "loop-stem" structure as a minimal requirement. To examine the role of the consensus uridine residue at position 8 (U8) adjacent to the D "loop-stem" region in the recognition of Gm-methylase, 4-thiouridine at this position (s4U8) in Escherichia coli tRNAfMet was modified reversibly with S-benzylthioisothiourea (sBTIU) or irreversibly by UV light. The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with beta-mercaptoethanol. Although both of the modified tRNAfMetS showed larger Km (although to different extents) and slightly smaller Vmax than the intact tRNAfMet, they retained full activities of methylation with tRNA(adenine-1-)-methyltransferase (m1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from T. thermophilus strain HB 27. The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNAfMetS showed methylation efficiency (Vmax/Km) not appreciably different from that of the unmodified 5'-half fragment. These results suggest that the conformation of S4U8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.  相似文献   

9.
10.
The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNA(Met) is closely related to the imported elongator tRNA(Met). Thus, the distinct localization of the two tRNAs(Met) must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAs(Met). Furthermore, it was shown that the tRNA(Met) T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNA(Met) indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAs(Met) into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNA(Met).  相似文献   

11.
Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).  相似文献   

12.
An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.  相似文献   

13.
14.
By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine.  相似文献   

15.
16.
The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Delta trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA(Met)(f)). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA(Met)(f) and tRNA(Phe). High pressure liquid chromatography analysis showed the methylated product to be m(1)G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1-33 (Delta1-33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Delta1-33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m(1)G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.  相似文献   

17.
A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.  相似文献   

18.
Transfer RNAs (tRNAs) play important roles to decode the genetic information contained in mRNA in the process of translation. The tRNA molecules possess conserved nucleotides at specific position to regulate the unique function. However, several nucleotides at different position of the tRNA undergo modification to maintain proper stability and function. The major modifications include the presence of pseudouridine (Ψ) residue instead of uridine and the presence of m5-methylation sites. We found that, Ψ13 is conserved in D-stem, whereas Ψ38 & Ψ39 were conserved in the anti-codon loop (AL) and anti-codon arm (ACA), respectively. Furthermore, Ψ55 found to be conserved in the Ψ loop. Although, fourteen possible methylation sites can be found in the tRNA, cyanobacterial tRNAs were found to possess conserved G9, m3C32, C36, A37, m5C38 and U54 methylation sites. The presence of multiple conserved methylation sites might be responsible for providing necessary stability to the tRNA. The evolutionary study revealed, tRNAMet and tRNAIle were evolved earlier than other tRNA isotypes and their evolution is date back to at least 4000 million years ago. The presence of novel pseudouridination and m5-methylation sites in the cyanobacterial tRNAs are of particular interest for basic biology. Further experimental study can delineate their functional significance in protein translation.  相似文献   

19.
K Marcu  D Marcu    B Dudock 《Nucleic acids research》1978,5(4):1075-1092
An unusual class of wheat germ tRNAs has been isolated which completely lacks ribothymidine (rT) and contains an unmodified uridine in its place. We discuss here the isolation, identification and properties of these tRNAs. The rT-lacking tRNAs of wheat germ are essentially limited to the glycine isoacceptors (a minimum of five identifiable species), three threonine and at least, one tyrosine tRNA. All tRNAs were obtained 70-100% pure by chromatographic methods, and were detected by their ability to be methylated by E. coli rT-forming uracil methyltransferase with methyl-labeled S-adenosyl-L-methionine (SAM) as the methyl donor. In vitro methylation of each of the tRNAs resulted in the formation of 1 mole of rT per mole of tRNA. In the one case analyzed in detail (tRNA1Gly), all of the rT was found to be located at the 23rd position from the 3' end of the tRNA molecule. Following complete digestion of four highly purified glycine isoacceptors (tRNAGly1,4,5,6) to nucleosides and subsequent periodate oxidation and 3H potassium borohydride reduction, all were found to contain an unusually high level of 5-methylcytidine (m5C) (3-4 residues per molecule), and all contained no rT. The possible correlation between the presence of m5C and the absence of rT is discussed. All of the chromatographically purified glycine tRNAs function in a wheat germ cell-free protein synthesizing system and polymerize glycine in response to either poly G or poly (G, U).  相似文献   

20.
In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.  相似文献   

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