The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.     

Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs.     

Rodent type 2 Alu family,rat identifier sequence,rabbit C family,and bovine or goat 73-bp repeat may have evolved from tRNA genes

Summary Close structural resemblances between several mammalian highly or moderately repetitive families and some specific tRNAs were detected. The rodent type 2 Alu family, rat identifier (ID) sequences, rabbit C family, and bovine or goat 73-bp repeat are most homologous with lysine tRNA₅, phenylalanine tRNA, glycine tRNA, and glycine tRNA, respectively. The homologies extend to secondary structures, and the homologous nucleotides are located on nearly the same secondary structures. The repetitive families mentioned have a common structural organization, with a tRNA-like sequence devoid of an aminoacyl stem region. These features suggest that these repetitive families may be generated by nonhomologous recombination between a tRNA gene and a tRNA-unrelated block.     

The nucleotide sequence of arginine tRNACCG from bovine liver

The nuclotide sequence of arginine tRNA(CCG) from bovine liver was determined to be: pG-A-C-C-C-A-G-U-m(1)G-m(2)G-C-C-U-A-A-D-Gm-G-A-D-A-A-G-G-C-A-psi-C-A-G-C-Cm-U-C-C-G-m(1)G-A-G-C-U-G-G-G-G-A-D-U-G-psi-G-G-G-T-psi-C-G-m(1)A-G-U-C-C-C-A-U-C-U-G-G-G-U-C-G-C-C-A(OH). This arginine tRNA is 76 nucleotides in length with 13 modified bases and has an anticodon of CCG. The sequence of this molecule is substantially different from those of other arginine tRNAs sequenced to date and is the only arginine tRNA sequenced which would be expected to recognize the codon CGG.Images     

The nucleotide sequence of a glutamate tRNA from rat liver.

A glutamate tRNA from rat liver was purified. By means of post-labeling techniques, its nucleotide sequence was shown to be: pU-C-C-C-A-C-A-U-m1G-G-U-C-psi-A-G-C- G-G-D-D-A-G-G-A-U-U-C-C-U-G-G-psi-U-mcm5s2U-U-C-A-C-C-C-A-G-G-C-G- G-C-m5C-m5C-G-G-G-Tm-psi-C-G-A-C-U-C-C-C-G-G-U-G-U-G-G-G-A-A-C-C-AOH. The sequence is remarkably similar to that of tRNA4Glu from Drosophila melanogaster. Only 10 out of 75 nucleotides in the two tRNAs are different.     

Nucleotide sequence of starfish initiator tRNA.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.     

The nucleotide sequence of a glutamine tRNA from rat liver.

A glutamate tRNA from rat liver was purified. By means of post-labeling techniques, its nucleotide sequence was shown to be: pU-C-C-C-A-C-A-U-m1G-G-U-C-psi-A-G-C- G-G-D-D-A-G-G-A-U-U-C-C-U-G-G-psi-U-mcm5S2U-U-C-A-C-C-C-A-G-G-C-G- G-C-m5C-m5C-G-G-G-Tm-psi-C-G-A-C-U-C-C-C-G-G-U-G-U-G-G-G-A-A-C-C-AOH. The sequence is remarkably similar to that of tRNAGlu from Drosophila melanogaster. Only 10 out of 75 nucleotides in the two tRNAs are different.     

Nucleotide sequence of salmon testes and salmon liver cytoplasmic initiator tRNA

Initiator tRNAs from the cytoplasm of salmon testes and salmon liver have been purified. The nucleotide sequence of these initiator tRNAs has been determined and found identical to that of initiator tRNA from mammalian cytoplasm. The only difference is the extent of modification of the nucleoside located between the dihydrouridine and the anticodon stems. In the salmon tRNAs, this modified nucleoside is predominantly N²N²-dimethyl guanosine, whereas in the mammalian initiator tRNA it is N²-methyl guanosine.     

Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7

The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism. However, the actual cleavage site in the splicing process was not determined for 13 interrupted tRNAs because of the presence of the same nucleotides at both 5′ and 3′ border regions of each intron. The cleavage sites for all the introns, which were determined by an in vitro cleavage experiment with recombinant splicing endonuclease as well as cDNA sequencing of the spliced tRNAs, indicated that non-canonical BHB structure motifs were also recognized and processed by the splicing machinery in this organism. This is the first report to empirically determine the actual cleavage and splice sites of introns in the whole set of archaeal tRNA genes, and reassigns the exon-intron borders with a novel and more plausible non-canonical BHB structure.     

Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila.

The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques. The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH. The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No. 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms. The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.     

Gram-scale purification of arginine, formylmethionione, glutamic acid and the nylalanine transfer ribonucleic acids from E. coli K-12MO7

Gram-sized quantities of purified arginine, formylmethionine, glutamic acid, and phenylalanine-2 tRNAs have been prepared from pools of E. coli K–12 MO7 mixed tRNAs by reversed-phase chromatography after preliminary fractionation on DEAE-cellulose. Purified formylmethionine tRNA and partially purified arginine tRNA and glutamie acid tRNA were obtained from large-scale RPC–3 runs (4 × 36 in. column). The arginine tRNA was further purified by rechromatography on RPC–4 columns, and the gluatmic acid tRNA by rechromatography on an RPC–3 column. Two phenylalanine tRNAs were resolved on large-scale (2 × 96 in. column) RPC–3 runs; only the second phenylalanine tRNA reached a satisfactory degree of activity. About 0.88 g of arginine tRNA, 70% activity; 3.32 g of formylmethionine tRNA, 97% activity; 0.80 g of glutamic acid tRNA, 83% activity and O.92 g of phenylalanine-2 tRNA, 78% activity, were produced. The processing steps employed are reliable and reproducible and the procedure is amenable to routine production of these tRNAs.     

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.     

Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila

Characterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced.     

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.     

Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.     

A novel cloverleaf structure found in mammalian mitochondrial tRNA(Ser) (UCN).

Bovine mitochondrial tRNA(Ser) (UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser) (UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser) (UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser) (UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser) (UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser) (UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.