Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast.

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.

Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae.

To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested. The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs. Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity. Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates. These results demonstrate that the protein encoded by the S. cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31. Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p. Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.

Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p.

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA.

So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability.

RNA:pseudouridine synthetase Pus1 from Saccharomyces cerevisiae: oligomerization property and stoichiometry of the complex with yeast tRNA(Phe).

Yeast RNA:pseudouridine synthetase Pus1 catalyzes the formation of pseudouridines in tRNAs. We report here the quaternary structure of purified recombinant Pus1 in solution. At low concentration, in the absence of tRNA, Pus1 oligomerizes while at high concentration it precipitates. This oligomerization/aggregation can be prevented by addition of dodecyl-beta-D-maltoside or of yeast tRNA(Phe). The detergent does not significantly interfere with substrate binding or with activity of Pus1. The stoichiometry of the Pus1/tRNA(Phe) complex is 1/1. We conclude that the detergent covers an hydrophobic region of the RNA binding pocket responsible for Pus1 aggregation.

Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus.

The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli. The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps. The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours). pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe). It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups. Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product. The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions. Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast. The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule. These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group. In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule.

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w.

Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNA(Ala) are major epitopes for anti-PL-12 myositis autoantibodies.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.

Pseudouridine synthetase Pus1 of Saccharomyces cerevisiae: kinetic characterisation, tRNA structural requirement and real-time analysis of its complex with tRNA.

Pseudouridine synthetase Pus1 from Saccharomyces cerevisiae is a multisite-specific enzyme that catalyses the formation of pseudouridine residues at different positions in several tRNA transcripts. Recombinant Pus1, tagged with six histidine residues at its N terminus was expressed in Escherichia coli and purified. Transcripts of yeast tRNAValand intronless yeast tRNAIlewere used as substrates to measure pseudouridine formation at position 27. The catalytic parameters Kmand kcatfor tRNAValand tRNAIlewere 420(+/-100) nM and 0.4(+/-0.1) min-1, 740(+/-100) nM and 0.5(+/-0.1) min-1, respectively. Pus1 possesses a general affinity for tRNA, irrespective of whether they are substrates. Its equilibrium dissociation constant ranges from 15 nM for the substrate yeast tRNAValand non-substrate yeast intronless tRNAPhe, to 150 nM for the substrate yeast intronless tRNAIle. The difference in the affinity for the different tRNA species is not reflected in the specific activity of the enzyme, indicating that the binding of Pus1 to tRNA is not the kinetically limiting step. The importance of tertiary base-pairs was investigated with several variants of yeast tRNAs. Although dispensable for activity, both the presence of a D-stem-loop and the presence of a G26.A44 base-pair, near the target uridine U27, are important elements for Pus1 tRNA high affinity recognition. The presence of a G26.A44 base-pair in tRNA increases its association constant rate with Pus1 (ka) by a factor of approximately 100, resulting in a decrease of the overall equilibrium dissociation constant (Kd). The dissociation rate (kd) is the same, independent of the presence of a G26.A44 base-pair in the tRNA. A model describing the interaction of Pus1 with tRNA is proposed.

Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro.

The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet). The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57. Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli.

The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF. Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa). The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.

Pseudouridine and ribothymidine formation in the tRNA-like domain of turnip yellow mosaic virus RNA.

The last 82 nucleotides of the 6.3 kb genomic RNA of plant turnip yellow mosaic virus (TYMV), the so-called 'tRNA-like' domain, presents functional, structural and primary sequence homologies with canonical tRNAs. In particular, one of the stem-loops resembles the TPsi(pseudouridine)-branch of tRNA, except for the presence of a guanosine at position 37 (numbering is from the 3'-end) instead of the classical uridine-55 in tRNA (numbering is from the 5'-end). Both the wild-type TYMV-RNA fragment and a variant, TYMV-mut G37U in which G-37 has been replaced by U-37, have been tested as potential substrates for the yeast tRNA modification enzymes. Results indicate that two modified nucleotides were formed upon incubation of the wild-type TYMV-fragment in a yeast extract: one Psi which formed quantitatively at position 65, and one ribothymidine (T) which formed at low level at position U-38. In the TYMV-mutant G37U, besides the quantitative formation of both Psi-65 and T-38, an additional Psi was detected at position 37. Modified nucleotides Psi-65, T-38 and Psi-37 in TYMV RNA are equivalent to Psi-27, T-54 and Psi-55 in tRNA, respectively. Purified yeast recombinant tRNA:Psisynthases (Pus1 and Pus4), which catalyze respectively the formation of Psi-27 and Psi-55 in yeast tRNAs, are shown to catalyze the quantitative formation of Psi-65 and Psi-37, respectively, in the tRNA-like 3'-domain of mutant TYMV RNA in vitro . These results are discussed in relation to structural elements that are needed by the corresponding enzymes in order to catalyze these post-transcriptional modification reactions.

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity.

We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.

Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition.

RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant Pus1, expressed in Escherichia coli, contains one atom of zinc per 63-kDa monomer, as determined by atomic absorption spectroscopy. This zinc ion could not be removed by treatment with EDTA or urea. However, a zinc-depleted enzyme was obtained after prolonged dialysis against the specific chelating agent 1,10-phenanthroline. Removal of the zinc ion resulted in inactivation of the enzyme with concomitant loss of its ability to bind tRNA. Dialysis of the zinc-depleted inactive enzyme against buffer containing zinc ions led to recovery of up to 25% of bound zinc in parallel with 25% of its initial activity. Removal of the tightly bound zinc atom resulted in a conformational change of the protein, as determined by analytical ultracentrifugation, with minor changes in the internal structure of the protein, as evidenced by circular dichroism and infrared and fluorescence spectroscopy. Our results are consistent with a structural role for the zinc in the tRNA-pseudouridine synthetase Pus1; zinc ion could maintain the association between domains structurally organized around the coordinated metal ion. Zinc chelation was never demonstrated for any of the tRNA-pseudouridine synthetases characterized so far.

Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop.

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).

Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: an in vivo study with Xenopus laevis oocytes.

We have investigated the specificity of the eukaryotic enzymatic machinery that transforms adenosine at position 37 (3' adjacent to anticodon) of several tRNAs into threonylcarbamoyladenosine (t6A37). To this end, 28 variants of yeast initiator tRNAMet and yeast tRNAVal, devoid of modified nucleotide, were produced by in vitro transcription with T7 polymerase of the corresponding synthetic tRNA genes and microinjected into the cytoplasm of Xenopus laevis oocytes. Threonylcarbamoyl incorporation was analyzed in tRNA transcripts mutated in the anticodon loop by substitution, deletion, or Insertion of nucleotides, or in the overall 3D structure of the tRNA by altering critical tertiary interactions. Specifically, we tested the effects of altering ribonucleotides in the anticodon loop, changes of the loop size, perturbations of the overall tRNA 3D structure due to mutations disruptive of the tertiary base pairs, and truncated tRNAs. The results indicate that, in addition to the targeted A37, only U36 was absolutely required. However, A38 in the anticodon loop considerably facilitates the quantitative conversion of A37 into t6A37 catalyzed by the enzymes present in X. laevis. The anticodon positions 34 and 35 were absolutely "neutral" and can accept any of the four canonical nucleotides A, U, C, or G. The anticodon loop size may vary from six to eight nucleotides, and the anticodon stem may have one mismatch pair of the type AxC or GxU at location 30-40 without affecting the efficiency of t6A37 formation and still t6A37 is efficiently formed. Although threonylcarbamoylation of A37 occurred with tRNA having limited perturbations of 3D structure, the overall L-shaped architecture of the tRNA substrate was required for efficient enzymatic conversion of A37 to t6A37. These results favor the idea that unique enzymatic machinery located in the oocyte cytoplasm catalyzes the formation of t6A37 in all U36A37-containing tRNAs (anticodon NNU). Microinjection of the yeast tRNAMeti into the cytoplasm of X. laevis oocytes also revealed the enzymatic activities for several other nucleotide modifications, respectively m1Gg, m2G10, m(2)2G26, m7G46, D47, m5C48/49, and m1A58.

A pseudoknotted tRNA variant is a substrate for tRNA (cytosine-5)-methyltransferase from Xenopus laevis.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

Transfer RNA recognition by the Escherichia coli delta2-isopentenyl-pyrophosphate:tRNA delta2-isopentenyl transferase: dependence on the anticodon arm structure.

To elucidate the sequence elements required in the anticodon stem for the recognition of Escherichia coli tRNA(Ser) (GGA) by the E. coli isopentenyl-tRNA:A37 transferase (IPTT), which result in the conversion of A37 into isopentenylated i6A37, we have tested and characterized in vitro T7-runoff transcripts of 17 variants of E. coli tRNA(Ser)(GGA) and 7 other tRNAs from E. coli and yeast. Our results indicate that, instead of a stringent specific anticodon stem and loop sequence, the key feature required for the recognition of E. coli tRNAs by IPTT is the A36A37A38 sequence occurring within the seven-membered anticodon loop, and the retention of the standard helical structure and flexibility, especially in the proximal anticodon stem. The G30*U40 mismatch base pair close to the anticodon loop is strictly avoided. The frequent occurrence of a C-G base pair in the three stem locations closest to the loop (positions 29-41, 30-40 and 31-39) or the occurrence of even one such C-G base pair along with some other similarly less suited, but individually tolerated deviations can also totally abolish the A37 isopentenylation of tRNA. For the position 30-40, the G-C base pair is shown uniquely suited, whereas for the adjoining 29-41 stem location, a purine-pyrimidine base pair with pyrimidine on the 3'-side is strongly preferred. Retention of the overall 3D tRNA structure is favorable for isopentenylation and allows some tolerance of proximal stem sequence deviations. Our data suggest a recognition mode that implies the interaction of IPTT with the strictly conserved A36A37A38 sequence and the other functional groups located in the minor groove of the anticodon stem.