The Origin of tRNA Deduced from Pseudomonas aeruginosa 5' Anticodon-Stem Sequence : Anticodon-stem loop hypothesis.

The riddle of the origin of life is unsolved as yet. One of the best ways to solve the riddle would be to find a vestige of the first life from databases of DNA and/or protein of modern organisms. It would be, especially, important to know the origin of tRNA, because it mediates between genetic information and the amino acid sequence of a protein. Here I attempt to find a vestige of the origin and evolution of tRNA from base sequences of Pseudomonas aeruginosa tRNA gene. It was first perceived that 5' anticodon (AntiC) stem sequences of P. aeruginosa tRNA for translation of G-start codon (GNN) are intimately and mutually related. Then, mutual relations among all of the forty-two 5' AntiC stem sequences of P. aeruginosa tRNA were examined. These relationships imply that P. aeruginosa tRNA originated from four anticodon stem-loops (AntiC-SL) translating GNC codons to the corresponding four amino acids, Gly, Ala, Asp and Val (where N is G, C, A, or T). In contrast to the case of AntiC-stem sequence, a mutual relation map could not be drawn with D-, T- and acceptor-stem sequences of P. aeruginosa tRNA. Thus I conclude that the four AntiC-SLs were the first primeval tRNAs.

7-Methylguanosine at the anticodon wobble position of squid mitochondrial tRNA(Ser)GCU: molecular basis for assignment of AGA/AGG codons as serine in invertebrate mitochondria.

In mitochondria of the squid, Loligo bleekeri, both the AGA and AGG codons are considered to correspond to serine instead of arginine as in the universal genetic code, and its genome encodes a single tRNA(Ser) gene with the anticodon GCT. Therefore, this gene product, tRNA(Ser)GCU, should be able to translate all four AGN (N; U, C, A, and G) codons as serine. To elucidate this recognition mechanism, the tRNA(Ser)GCU was isolated from squid liver and its complete nucleotide sequence determined. The tRNA(Ser)GCU was found to possess 7-methylguanosine (m7G) at the wobble position of the anticodon. This suggests that in the squid mitochondrial system, tRNA(Ser)GCU with the anticodon m7GCU can recognize not only the usual serine codons AGU and AGC, but also the unusual serine codons AGA and AGG, as in the case of starfish mitochondria (Matsuyama et al., J. Biol Chem. 273 (1988) 3363-3368).

Eukaryotic tRNAs(Pro): primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon.

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2'-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo.

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.

Identification of a modified nucleoside located in the first position of the anticodon of Torulopsis utilis tRNAPro as 5-carbamoylmethyluridine.

An unknown nucleoside in the first position of the anticodon of Torulopsis utilis tRNAPro has been isolated. The UV, 1H NMR and secondary ion mass spectra indicated that this nucleoside is a uridine derivative, 5-carbamoylmethyluridine. The structure was completely established by comparison of the instrumental analysis results and chromatographic behavior of the isolated nucleoside with those of a synthetic sample.

Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly.

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.

Modified nucleoside, 5-carbamoylmethyluridine, located in the first position of the anticodon of yeast valine tRNA.

A novel modified nucleoside located in the first position of the anticodon of yeast tRNAVal2a was isolated and its chemical structure was characterized as 5-carbamoylmethyluridine by means of ultraviolet absorption spectrum, mass spectrum, and nuclear magnetic resonance spectrum.

A selenium-containing nucleoside at the first position of the anticodon in seleno-tRNAGlu from Clostridium sticklandii.

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.

Purification and properties of bovine liver seryl-tRNA synthetase.

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on Matrex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87 000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr, was estimated as 170 000 for the dimeric native enzyme (alpha 2 type) by chromatography on Sephacryl S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 microM, respectively. The Km values for tRNASerIGA and tRNASerCmCA were 1.40 microM and 1.25 microM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase.

Minor serine tRNA containing anticodon NCA (C4 RNA) from human and mouse cells.

The nucleotide sequence of C4 RNA, one of the "4.5S RNAs" of HeLa cells, was determined. This RNA consists of 90 nucleotides containing C-C-A at its 3'-terminus. The sequence can be drawn to form a clover-leaf structure with several unusual features and with anticodon NCA. The short term labeled molecule contains triphosphates at its 5'-terminus, whereas the mature molecule contains only monophosphate. Therefore, there must be no precursor nucleotide at the 5'-end in the primary transcript of this tRNA. Mouse cells also contain C4 RNA. Only one base exchange was observed in the extra arm in human and mouse C4 RNAs. The molecule purified from mouse liver showed serine acceptor ability.

A modified nucleoside located in the anticodon of proline tRNA from Torulopsis utilis is 5-carbamoylmethyluridine.

A modified nucleoside has been isolated from the first position of the anticodon of Torulopsis utilis tRNAPro. It was identified to be an uridine derivative, 5-carbamoylmethyluridine from analyses of its UV, 1H-NMR, and secondary ion mass spectra.

Novel features in the genetic code and codon reading patterns in Neurospora crassa mitochondria based on sequences of six mitochondrial tRNAs.

We report the sequences of Neurospora crassa mitochondrial alanine, leucine(1), leucine(2), threonine, tryptophan, and valine tRNAs. On the basis of the anticodon sequences of these tRNAs and of a glutamine tRNA, whose sequence analysis is nearly complete, we infer the following: (i) The N. crassa mitochondrial tRNA species for alanine, leucine(2), threonine, and valine, amino acids that belong to four-codon families (GCN, CUN, ACN, and GUN, respectively; N = U, C, A, or G) all contain an unmodified U in the first position of the anticodon. In contrast, tRNA species for glutamine, leucine(1), and tryptophan, amino acids that use codons ending in purines (CA(G) (A), UU(G) (A), and UG(G) (A), respectively) contain a modified U derivative in the same position. These findings and the fact that we have not detected any other isoacceptor tRNAs for these amino acids suggest that N. crassa mitochondrial tRNAs containing U in the first position of the anticodon are capable of reading all four codons of a four-codon family whereas those containing a modified U are restricted to reading codons ending in A or G. Such an expanded codon-reading ability of certain mitochondrial tRNAs will explain how the mitochondrial protein-synthesizing system operates with a much lower number of tRNA species than do systems present in prokaryotes or in eukaryotic cytoplasm. (ii) The anticodon sequence of the N. crassa mitochondrial tryptophan tRNA is U(*)CA and not CCA or CmCA as is the case with tryptophan tRNAs from prokaryotes or from eukaryotic cytoplasm. Because a tRNA with U(*)CA in the anti-codon would be expected to read the codon UGA, as well as the normal tryptophan codon UGG, this suggests that in N. crassa mitochondria, as in yeast and in human mitochondria, UGA is a codon for tryptophan and not a signal for chain termination. (iii) The anticodon sequences of the two leucine tRNAs indicate that N. crassa mitochondria use both families of leucine codons (UU(A) (G) and CUN; N = U, C, A, or G) for leucine, in contrast to yeast mitochondria [Li, M. & Tzagoloff, A. (1979) Cell 18, 47-53] in which the CUA leucine codon and possibly the entire CUN family of leucine codons may be translated as threonine.