The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron.

The maize chloroplast gene encoding tRNA Leu UAA has been sequenced. It contains a 458 base pair intron between the first and second bases of the anticodon. The tRNA is 88 nucleotides long (the 3'-terminal CCA sequence included which, however, is not encoded by the gene) and differs in only four nucleotides (modified nucleotides are not considered) from the corresponding isoacceptor from bean chloroplasts. The unusual position of the intron in this maize chloroplast tRNA gene suggests a splicing model different from that generally accepted for eukaryotic split tRNA genes.     

Modeling the tertiary interactions in the eukaryotic selenocysteine tRNA.

A novel three-dimensional model of tertiary interactions in the core region of the eukaryotic selenocysteine tRNA is proposed based on the analysis of available nucleotide sequences. The model features the 7/5 tRNA(Sec) secondary structure characterized by seven and five base pairs in the acceptor and T-stems, respectively, and four nucleotides in the connector region between the acceptor and D-stems. The model suggests a unique system of tertiary interactions in the area between the major groove of the D-stem and the first base pair of the extra arm that provides a rigid orientation of the extra arm and contributes to the overall stability of the molecule. The model is consistent with available experimental data on serylation, selenylation, and phosphorylation of different tRNA(Sec) mutants. The important similarity between the proposed model and the structure of the tRNA(Ser) is shown. Based on this similarity, the ability of some tRNA(Ser) mutants to be serylated, selenylated, and phosphorylated was evaluated and found to be in a good agreement with experimental data.     

The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition

Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.     

Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu 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 tRNA₁^met 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.     

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.     

A phase relationship associates tRNA structural gene sequences with nucleosome cores

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.     

Lysine tRNAs from rat liver: lysine tRNA sequences are highly conserved

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

Strong magnesium binding sites in yeast phenylalanine transfer RNA.

To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op....Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m2G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it links the dihydrouridine arm with the extra (V) loop.     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells

Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.