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1.
We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNAMet but not for yeast tRNAMet i or E. coli tRNAMet f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNAMet i susceptible to the specific cleavage reaction. Using crystallographic data of tRNAMet f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNAMet i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.  相似文献   

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Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo5UAC) contains a unique modification, N6-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA1Val. Inactivation of yfiC gene abolishes m6A formation in tRNA1Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA1Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m6A in tRNA1Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.  相似文献   

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Primary structure of Bacillus subtilis tRNAsTyr   总被引:4,自引:0,他引:4  
tRNAITyr and tRNAIITyr have been purified from B.subtilis and their nucleotide sequence determined. tRNAITyr differs from tRNAIITyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i6A and ms2i6A respectively.  相似文献   

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Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNAPhe forms a trinucleotide loop in solution, but Mg2+ and dimethylallyl modification of A37 N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ32, ψ39, and A37 C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ32 on the anticodon stem-loop from E.coli tRNAPhe. The ψ32 modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ32-A38 and U33–A37 and the base of ψ32 stacks between U33 and A31. The glycosidic bond of ψ32 is in the anti configuration and is paired with A38 in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ32 modification increases the melting temperature of the stem by ~3.5°C, although the ψ32 and U33 imino resonances are exchange broadened. The results suggest that ψ32 functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.  相似文献   

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Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes. Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37th position and it is next to the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m1G37) methyltransferase (TrmD). It is deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37th and 38th position except three tRNA subsets having G residues at 36th and 39th positions. Therefore we propose that m1G37 modification may be feasible at 36th, 37th, 38th, 39th and 40th positions next to the anticodon of tRNAs. Collectively, methylation at G residues close to the anticodon may be possible at different positions and without restriction of anticodon 3rd base A, C, U or G.  相似文献   

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Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNATyr G A and elongator tRNAMet CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNATyr. Here we have studied the expression of an Arabidopsis elongator tRNAMet gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNAMet precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNAMet to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNAMet splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNAMet splicing and that a highly structured intron is indispensable for pre-tRNAMet splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNAMet gene, is efficiently processed and spliced in both plant extracts.  相似文献   

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Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg2+ and Mn2+ to yeast tRNATyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNATyr, spin labeled at the hypermodified purine (i6A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNATyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNATyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNATyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i6A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNATyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn2+ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.  相似文献   

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The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA1leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA1met and su+III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.  相似文献   

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tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1+ and tit1-Δ cells by using a β-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNACys at a cognate codon and that of tRNATyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at noncognate codons. S. pombe cells lacking tit1+ exhibit slow growth in glycerol or rapamycin. While existing data link wobble base U34 modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37 was unknown. Indeed, we found a biased presence of i6A37-cognate codons in high-abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirmed the decreased translational efficiency of mRNAs in tit1-Δ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.  相似文献   

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The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U38 and U39 in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ38 or Ψ39 is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNAGln(UUG), which normally has Ψ38. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s2U moiety of mcm5s2U34 of tRNAGln(UUG) and the other two cytoplasmic species with mcm5s2U34, that the reduced s2U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s2U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ38,39 provides evidence that Ψ38 is important for function of tRNAGln(UUG) at permissive temperature, and indicates that Ψ39 is important for the function of tRNATrp(CCA) in trm10Δ pus3Δ mutants and of tRNALeu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ38 and Ψ39 in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.  相似文献   

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《FEBS letters》1986,202(1):12-18
The digestion of yeast initiator methionine tRNA with mung bean nuclease and U2 ribonuclease yielded 5'- and 3'-fragments, respectively. These two fragments together represent the entire tRNA sequence except for A35, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C35 was reformed. Subsequent treatment of this product with CCA-transferase yielded a full-length methionine tRNA having an arginine CCU anticodon. This recombinant tRNAMet (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNAMet (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.  相似文献   

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