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PubMed1975

Biochemical research on oogenesis. Comparison between transfer RNAs from somatic cells and from oocytes in Xenopus laevis.

The properties of tRNA from oocytes of Xenopus laevis were compared with those of tRNA from somatic cells of the same species. Both types of tRNAs were found to have the same average length and to contain an equal variety of modified nucleotides. However, tRNA from small oocytes differed from somatic tRNA by its chromatographic behavior on methylated-albumin kieselguhr columns. The elution profiles from reversed-phase chromatography 5 columns of several aminoacyl tRNAs were compared after charging somatic and oocyte tRNSs with 3H or 14C-labelled amino acids. Striking differences in peak position were observed when tRNA from small oocytes was mixed and co-chromatographed with somatic tRNA. The differences were less important when tRNAs from large oocytes and from somatic cells were compared. Mixtures of egg and somatic tRNAs gave completely or almost completely coincident elution profiles. Only one isoacceptor, tNRA1Met (initiator tRNA) had the same position in all reversed-phase chromatography 5 chromatograms. The results are discussed in terms of possible post-transcriptional modifications of tRNA in the course of oogenesis. An alternative explanation resorting to changes in the tRNA population of the growing oocyte is also envisaged.

Animals↗
PubMed1992

Structural requirements for tRNA methylation. Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNA(1Leu) structural variants.

The Escherichia coli enzyme tRNA(m1G)methyltransferase, one of a group of post-transcription tRNA-modifying enzymes, shows remarkable specificity in selecting the tRNA species and the specific guanosine base to be methylated. To examine the structural basis of this specificity, we synthesized a total of 15 modifications of tRNA(1Leu) and measured their methylation reaction kinetics in vitro. Elimination of any one of the three tRNA side loops, the V loop, the T loop, or the D loop, reduced the Vmax for methylation by about 1 order of magnitude. Elimination of all three side loops reduced Vmax by about 2 orders of magnitude. Clearly, gross tRNA structure is important for full enzyme activity. At the bottom of the stem proximal to the anticodon loop, in the pair at positions 31-39, substitution of a G-C for a C-G, a change that should not weaken the helical structure, had little effect on Vmax or Km. However, substitution of a G for a C increased Vmax and Km, whereas substitution of a C for G sharply reduced Vmax and, to a lesser extent, Km. These results appear to be a consequence of the principle that purines are better than pyrimidines in the stacking of adjacent bases for stability. Stacking in the stem structure appears to be important for methylation enzyme activity. In the anticodon loop itself, changing a U to a C had little effect, but changing the G of the anticodon to a C reduced Vmax over 20-fold, demonstrating the importance of the presence of the anticodon G adjacent to the G being methylated for enzyme recognition.

Anticodon↗
PubMed1995

Influence of modified nucleosides on tRNA structure as probed by two plant nucleases.

Two new enzymatic probes have been used for structural investigations of native and unmodified transcript tRNA molecules. Both probes were single-strand-specific nucleases isolated from higher plants. The results obtained after enzymatic hydrolysis of tRNAs support the earlier hypothesis that posttranscriptional modifications in tRNA help to stabilize its structure and make it more rigid.

Base Sequence↗
PubMed2002

Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe).

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.

Alkyl and Aryl Transferases↗
PubMed1985

[Chemical modification of tryptophan residues of leucyl tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide].

The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed2001

A novel interaction of tRNA(Lys,3) with the feline immunodeficiency virus RNA genome governs initiation of minus strand DNA synthesis.

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)(4) sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

Base Sequence↗
PubMed1997

Essentially minimal sequence for substrate recognition by tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27.

Transfer RNA (guanosine-2'-)-methyltransferase (Gm-methylase, EC.2.1.1.32) from extreme thermophile, Thermus thermophilus HB27 is one of the tRNA-ribose modification enzymes; this enzyme specifically catalyze the transfer of a methyl group from S-adenosyl-L-methionine to 2'-OH of the ribose of the guanosine at position 18 in tRNA. A broad substrate specificity of Gm-methylase was observed using natural tRNAs as methyl group acceptors, which suggests that some local stractures common in tRNAs are recognized by the enzyme. By using yeast tRNA(Phe) variants obtained by transcription of their genes with T7 RNA polymerase, it was revealed that the residues G18 and G19, as well as the D-stem structure were primarily required for the methylation reaction and that the essentially minimal sequence for the substrate was Pyrimidine17-G18-G19. The other conserved sequences and the tertiary base-pairs were not essential, but G15, G46, U55 and C56 strongly affected the methylation efficiency.

Base Composition↗
PubMed1995

C to U editing and modifications during the maturation of the mitochondrial tRNA(Asp) in marsupials.

In marsupial mitochondria, the nucleotide residue at the second position of the anticodon of the tRNA for aspartic acid is changed post-transcriptionally such that the translational machinery recognizes it as a uracil rather than the cytosine residue encoded in the gene. By postlabeling nucleotide analysis, we show here that the cytosine residue is converted to a conventional uracil residue in an RNA editing event that affects approximately half of the tRNA molecules under steady state conditions. Furthermore, we have identified three different tRNA(Asp) species which all carry three pseudouridines and two methylations but have the anticodons GCC, GUC and QUC respectively, the latter representing a rare example of queuine incorporation into a mitochondrial tRNA. This allows us to describe a likely sequential order of modification of the tRNA(Asp), where methylations and conversions of uridines to pseudouridines precede the editing event, while the exchange of guanine by queuine takes place after the C to U editing event.

Animals↗
PubMed2005

The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells.

A substrate for protein kinase B (PKB)alpha in HeLa cell extracts was identified as methyltransferase-like protein-1 (METTL1), the orthologue of trm8, which catalyses the 7-methylguanosine modification of tRNA in Saccharomyces cerevisiae. PKB and ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27 in vitro. Ser27 became phosphorylated when HEK293 cells were stimulated with insulin-like growth factor-1 (IGF-1) and this was prevented by inhibition of phosphatidyinositol 3-kinase. The IGF-1-induced Ser27 phosphorylation did not occur in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deficient embryonic stem cells, but occurred normally in PDK1[L155E] cells, indicating that the effect of IGF-1 is mediated by PKB. METTL1 also became phosphorylated at Ser27 in response to phorbol-12-myristate 13-acetate and this was prevented by PD 184352 or pharmacological inhibition of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression of METTL1[S27D] or METTL1[S27E] did not rescue the growth phenotype of yeast lacking trm8. In contrast, expression of METTL1 or METTL1[S27A] partially rescued growth. These results demonstrate that METTL1 is inactivated by PKB and RSK in cells, and the potential implications of this finding are discussed.

Amino Acid Sequence↗
PubMed2005

Two different mechanisms for tRNA ribose methylation in Archaea: a short survey.

The biogenesis of tRNA involves multiple reactions including post-transcriptional modifications and pre-tRNA splicing. Among the three domains of life, only Archaea have two different mechanisms for tRNA ribose methylation: site-specific 2'-O-methyltransferases and C/D guided-RNA machinery. Recently, the first archaeal tRNA 2'-O-methyltransferase, aTrm56, has been characterized. This enzyme is found in all archaeal genomes sequenced so far except one and belongs to the SPOUT family (class IV) of RNA methyltransferases. Its substrate is the conserved C56 in the T-loop of archaeal tRNAs. In the crenarchaeon Pyrobaculum aerophylum, in which no homologue of this methyltransferase is found, a box C/D guide sRNP insures the ribose methylation of C56. Moreover, a new twist on tRNA processing is the finding, in most euryarchaeal tRNAtrp genes, of a box C/D guide RNA within their intron specifying methylation at two sites. Modification of tRNA is an integral part of the complex maturation process of primary tRNA transcripts. In addition to their role in modification, both modification enzymes and C/D guide RNPs may have a chaperone function insuring the precise folding of the mature, functional tRNA.

Archaea↗
PubMed1991

Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg).

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.

Affinity Labels↗
PubMed1988

Evidence for tertiary structure in natural single stranded RNAs in solution.

Binding isotherms (20 degrees C) of ethidium bromide to a number of tRNA species at various ionic strengths indicate that i) the number ni of intercalation sites is high 7 to 11 per molecule, in the low salt form III, but small, 2 to 1, at high Mg2+ or Na+ when form I predominates. ii) modification of tRNA at strategic positions for 3D folding prevents full expression of intercalation restriction iii) maximal restriction is obtained at salt concentrations higher than needed for full conversion to form I. It is inferred that restriction, which is not observed with bihelical RNA (or DNA), requires the native tRNA 3D structure but also some physical coupling between the region of 3D folding and bihelical arms. Ribosomal RNAs, some viral RNAs, mRNA from sheep mammary gland as well as the random copolymers Poly UG, Poly AUG, Poly AUCG all exhibit intercalation restriction. Hence 3D folding of the polyribonucleotide chains appears to be a feature common to single-stranded RNAs when free in solution under physiological conditions.

DNA↗
PubMed1995

Primary sequence and post-transcriptional modification pattern of an unusual mitochondrial tRNA(Met) from Tetrahymena pyriformis.

In a previous investigation of the rDNA region in Tetrahymena pyriformis mitochondrial DNA, we identified a putative tRNA(Met) gene [Heinonen et al. (1987) J. Biol. Chem. 262, 2879-2887]. On the basis of Northern hybridization analyses, we suggested that this gene is expressed, even though the resulting tRNA would be unusually small and have an atypical dihydrouridine stem-loop domain. We report here the complete nucleotide sequence and post-transcriptional modification pattern of this tRNA(Met), confirming its predicted primary structure and supporting the view that this structurally aberrant species functions in translation in T. pyriformis mitochondria.

Animals↗
PubMed1988

Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA.

Escherichia coli mia strains were shown to lack delta 2-isopentenylpyrophosphate transferase activity, the first step in the synthesis of the 2-methylthio derivative of 6-(delta 2-isopentenyl) adenosine (ms2i6A). A double mutant, rpsL (Smp) miaA, was streptomycin dependent. The wild-type miaA gene was cloned by selecting for lambda recombinant bacteriophage which eliminated the streptomycin-dependent phenotype and was subsequently recloned into plasmid vectors. The cloned miaA gene restored the ms2i6A modification to tRNA. The miaA gene mapped to 95 min on the E. coli map, and we propose the order mutL-miaA-hflA-purA.

Adenine↗
PubMed2001

A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

Base Sequence↗
PubMed2004

Uniform binding of aminoacylated transfer RNAs to the ribosomal A and P sites.

The association and dissociation rate constants of eight different E. coli aminoacyl-tRNAs (aa-tRNAs) for E. coli ribosomes programmed with mRNAs of defined sequences were determined. Identical association and dissociation rate constants were observed for all eight aa-tRNAs in both the ribosomal A and P sites despite substantial differences in tRNA sequence, the type of esterified amino acid, and posttranscriptional modifications. These results indicate that the overall binding of all aa-tRNAs to the ribosome is uniform. However, when either the esterified amino acid or the tRNA modifications were removed, binding was no longer uniform. These results suggest that differences in tRNA sequences and tRNA modifications have evolved to offset differential thermodynamic contributions of the esterified amino acid and the codon-anticodon interaction so that ribosomal binding of all aa-tRNAs remains uniform.

Escherichia coli↗
PubMed2006

Modified view of tRNA: stability amid sequence diversity.

In a recent issue of Molecular Cell, a report from the Phizicky lab challenges two commonly held notions regarding tRNAs: (1) tRNAs are stable molecules shielded from the machinery that cause other RNAs to have short half-lives, and (2) the many modifications found on tRNAs serve only subtle biological functions. Alexandrov et al. (2006) show that tRNA modifications can play an important role in tRNA stability, as without particular modifications, tRNA half-life can be reduced from hours to minutes by a new turnover pathway.

RNA Processing, Post-Transcriptional↗
PubMed1986

High-level overexpression, rapid purification, and properties of Escherichia coli tRNA nucleotidyltransferase.

The cloned Escherichia coli cca gene, described in the accompanying paper (Cudny, H., Lupski, J. R., Godson, G. N., and Deutscher, M. P. (1986) J. Biol. Chem. 261, 6444-6449), has been used to construct strains that overproduce tRNA nucleotidyltransferase, the enzyme that synthesizes the CCA terminus of tRNA. Strain UT481 (pEC4), which contains a 1.9-kilobase cca gene insert in plasmid pUC8, overproduces the enzyme by about 100-150-fold, probably under the control of the cca gene promoter. A second strain, containing a plasmid with a 1.5-kilobase insert, overproduces tRNA nucleotidyltransferase by about 650-fold, to a level of about 3-4% of the soluble cell protein. In this case, overexpression was dependent on the lac promoter of the plasmid. A rapid, two-step procedure was developed to purify large amounts of the enzyme from strain UT481 (pEC4) that was about 40% pure, free of ribonucleases, and suitable for use as a reagent for modification of tRNA molecules. Preparation of milligram quantities of homogeneous tRNA nucleotidyltransferase was accomplished by two further chromatographic steps. The structural and catalytic properties of this purified enzyme were similar to those from partially purified preparations previously described. The availability of large amounts of pure tRNA nucleotidyltransferase will not permit a variety of structural and functional studies of the enzyme that previously were not possible.

Cell Division↗