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.

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.

Codon:anticodon and anticodon:anticodon interaction: evaluation of equilibrium and kinetic parameters of complexes involving a g:u wobble.

In order to learn about the effect of the G:U wobble interaction we characterized to codon:anticodon binding between triplets: UUC, UUU and yeast tRNAPhe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNAGlu2, E. coli tRNALys (anticodons: mam5s2UUC, and mam5S2UUU, respectively) and tRNAPhe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20 degrees C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency.

Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes.

We have investigated whether unmodified yeast phenylalanine transfer RNA as well as one of its precursors containing an intron of nineteen nucleotides in the anticodon (pre-tRNA-Phe) can become substrates for selected tRNA modification enzymes present in a eukaryotic cell. This study was done by microinjecting into the cytoplasm of Xenopus laevis oocytes transcripts completely deprived of the naturally occurring modified nucleotides; these were obtained in vitro from appropriate synthetic genes under the control of bacteriophage T7 promoter. During the in vitro transcription, 32P labels were introduced with the guanosine triphosphate thus allowing easy detection of guanosine modifications in tRNA by two-dimensional chromatography after complete digestion into 5'-mononucleotides by nuclease P1. Results indicate that modifications occur on five guanosines (at positions 10, 26, 34, 37 and 46) in yeast tRNA-Phe and only on three guanosines (at 10, 26 and 46) in yeast precursor tRNA-Phe. These are the modifications expected from the known nucleotide sequences of naturally occurring Xenopus and yeast tRNA-Phe, i.e. N2-methyl-G10, N2,N2-dimethyl-G26, 2'-O-methyl-G34, N1-methyl-G37 or Y nucleoside-37 and N7-methyl-G46. The rates of modifications occurring in the two kinds of tRNA-Phe are faster in the intron-less tRNA-Phe than in the intron-containing tRNA-Phe. However quantitative modifications are only observed after as long as 75 h incubation in the oocytes.

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.

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.

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal.

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture.

Information is still quite limited concerning the structural requirements in tRNA molecules for their post-transcriptional maturation by base and ribose modification enzymes. To address this question, we have chosen as the model system yeast tRNAAsp that has a known three-dimensional structure and the in vivo modifying machinery of the Xenopus laevis oocyte able to act on microinjected tRNA precursors. We have systematically compared the modification pattern of wild-type tRNAAsp with that of a series of structural mutants (21 altogether) altered at single or multiple positions in the D-, T-and the anticodon branch, as well as in the variable region. The experimental system allowed us to analyze the effects of structural perturbations in tRNA on the enzymatic formation of modified nucleosides at 12 locations scattered over the tRNA cloverleaf. We found that the formation of m1G37 and psi 40 in the anticodon loop and stem and psi 13 in the D-stem, were extremely sensitive to 3D perturbations. In contrast, the formation of T54, psi 55 and m1A58 in the T-loop, m5C49 in the T-stem and m2G6 in the amino acid accepting stem were essentially insensitive to change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA domains. The formation of m2G26 at the junction between the anticodon and the D-stem, of Q34 and manQ34 in the anticodon loop were sensitive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recognition by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to their sensitivities to structural perturbations in tRNA, is proposed.

Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA.

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in [18O]water. This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast.

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression.

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.

Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs.

Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T54) and pseudouridine (psi 55). To identify the tRNA elements recognized by tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA(Asp) mutants were used. Some variants contained point mutations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the T psi-loop with only one G.C base-pair as stem (9-mer). All substrates (full-sized tRNA(Asp) and various minihelices) were produced in vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl-L-methionine as a methyl donor. The results indicate that the minimal substrate for enzymatic formation of psi 55 is a stem/loop structure with only four G.C base-pairs in the stem, while a longer stem is required for efficient T54 formation. None of the conserved nucleotides (G53, C56, A58 and C61) and U54 for psi 55 or U55 for T54 formation can be replaced by any of the other three canonical nucleotides. Yeast tRNA:m5uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. Interestingly, in a tRNA(Asp) variant in which the T psi-loop was permuted with the anticodon-loop, the new U32 and U33 residues derived from the T psi-loop were quantitatively converted to T32 and psi 33, respectively. Structural mapping of this variant with ethylnitrosourea confirmed that the intrinsic characteristic structure of the T psi-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a T psi-loop structure. These results demonstrate that a local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase.

Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes.

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.

2'-O-methylation and inosine formation in the wobble position of anticodon-substituted tRNA-Phe in a homologous yeast in vitro system.

Four variants of yeast tRNA-Phe in which the anticodon and 3'-adjacent nucleotide (GmAAY) have been replaced by synthetic tetranucleotides NAAG (where N is each of the four canonical nucleosides G, C, U or A) are substrates for a yeast tRNA modification enzyme which catalyses the S-adenosyl-L-methionine dependent formations of Gm-34, Cm-34, Um-34, Am-34 and Im-34 (where Nm represents a 2'-O-methylnucleoside and I inosine). The kinetics of these nucleosides-34 2'-O-methylations reveal that yeast tRNA-Phe with G-34 (the natural substrate) is less efficiently modified than variants of the same tRNA containing U-34 and C-34. The formation of Am-34 in the tRNA containing A-34 was found to be particularly inefficient. However, in this tRNA, we observed the formation of I-34 followed by a 2'-O-methylation (giving rise to Im-34). In the yeast in vitro system described here, inosine formation is not dependent on the addition of any cofactor including hypoxanthine; the mechanism of inosine formation in yeast tRNA might therefore be distinct from that found in higher eukaryotes.

Enzymatic formation of N2,N2-dimethylguanosine in eukaryotic tRNA: importance of the tRNA architecture.

In eukaryotic tRNA, guanosine at position 26 in the junction between the D-stem and the anticodon stem is mostly modified to N2,N2-dimethylguanosine (m2(2)G26). Here we review the available information on the enzyme catalyzing the formation of this modified nucleoside, the SAM-dependent tRNA (m2(2)G26)-methyltransferase, and our attemps to identify the parameters in tRNA needed for efficient enzymatic dimethylation of guanosine-26. The required identity elements in yeast tRNA for dimethylation under in vitro conditions by the yeast tRNA(m2(2)G26)-methyltransferase (the TRM1 gene product) are comprised of two G-C base pairs at positions G10-C25 and C11-G24 in the D-stem together with a variable loop of at least five nucleotides. These positive determinants do not seem to act via base specific interactions with the methyltransferase; they instead ensure that G26 is presented to the enzyme in a favorable orientation, within the central 3D-core of the tRNA molecule. The anticodon stem and loop is not involved in such an interaction with the enzyme. In a heterologous in vivo system, consisting of yeast tRNAs microinjected into Xenopus laevis oocytes, the requirements for modification of G26 are less stringent than in the yeast homologous in vitro system. Indeed, G26 in several microinjected tRNAs becomes monomethylated, while in yeast extracts it stays unmethylated, even after extensive incubation. Thus either the X laevis tRNA(m2(2)G26)-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G26-methylating activities, one for G26-monomethylation, and one for dimethylation of G26.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Structure in tRNA data.

260 sequences of tRNA are compared, after their classification into six categories: prokaryotic (83 sequences) and eukaryotic (83 sequences) elongators, prokaryotic (10 sequences) and eukaryotic (11 sequences), initiators, lower eukaryotic mitochondrial tRNA (53 sequences) and archaebacterial tRNA (20 sequences). Beside the presence of invariable and semi-invariable positions in tRNA, non-random base distribution in almost all positions is also evident; most of them being characteristic of each class of tRNA. Therefore, during evolution it would seem that selectional pressures do act in so called variable positions as well as invariant positions of tRNA molecules. Results are discussed in relation to possible restrictions depending on functional and/or structural constraints of tRNA.

Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: a review.

In eukaryotic cells, especially in yeast, several genes encoding tRNAs contain introns. These are removed from pre-tRNAs during the maturation process by a tRNA-specific splicing machinery that is located within the nucleus at the nuclear envelope. Before and after the intron removal, several nucleoside modifications are added in a stepwise manner, but most of them are introduced prior to intron removal. Some of these early nucleoside modifications are catalyzed by intron-dependent enzymes while most of the others are catalyzed in an intron-independent manner. In the present paper, we review all known cases where the nucleoside modifications were shown to depend strictly on the presence of an intron. These are pseudouridines at anticodon positions 34, 35 and 36 and 5-methylcytosine at position 34 of several eukaryotic tRNAs. One common property of the corresponding intron-dependent modifying enzymes is that their activities are essentially dependent on the local specific architecture of the pre-tRNA molecule that comprises the anticodon stem and loop prolonged by the intron domain. Thus introns clearly serve as internal (cis-type) RNAs that guide nucleoside modifications by providing transient target sites in tRNA for selected nuclear modifying enzymes. This situation may be similar to the recently discovered (trans-type) snoRNA-guided process of ribose methylations of ribosomal RNAs within the nucleolus of eukaryotic cells.

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.

The nucleotide sequence of mannosyl-Q-containing tRNAAsp from Xenopus laevis oocytes.

The nucleotide sequence of tRNAAsp from X. laevis oocytes was determined as being: (sequence in text) The tRNA is 75 nucleotides long. This sequence is very similar (75% to 97% identity) to all other eukaryotic tRNAAsp sequenced so far, except for the bovine liver tRNAAsp (32% identity). The relation between the presence of a mannosyl group on queuosine (Q) at position 34 and the nucleotide sequence of the anticodon loop is discussed.