tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.     

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.     

The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.     

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.     

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E. coli: effects on in vivo function.

The selenocysteine-inserting tRNA (tRNA(Sec)) of E. coli differs in a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.     

Sequence of a new tRNA(Leu)(U*AA) from brewer's yeast.

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).     

Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.     

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.     

Effect of threonylcarbamoyl modification (t6A) in yeast tRNA Arg III on codon-anticodon and anticodon-anticodon interactions. A thermodynamic and kinetic evaluation

The effect of N-[9-(beta-D-ribofuranosyl) purin-6-ylcarbamoyl]threonine (t6A) adjacent to anticodon U-C-U of yeast tRNA Arg III (where U is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide-directed binding of tRNA on ribosomes, (b) the ribosome-free trinucleotide binding to the anticodon, (c) the anticodon-anticodon binding test. The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNA Arg III with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A-G-A triplet, and (c) E. coli tRNA Ser V (anticodon G-G-A). We therefore conclude that the stabilization effect of t6A occurs on U x A and U x G base pairs adjacent to the 5' side of the modified nucleoside, most probably by stacking.     

Primary structure of tRNA1ser from bovine liver

The primary structure of tRNA(1Ser) from the bovine liver has been studied. pG- A-C-G-A-G-G-U-G-G-C-ac4C-G-A-G-D-Gm-G-D-D-A-A-G-G- C-m2(2)-G-A-psi-G-G-A-m3C-U-G-C-U-A*-A-psi-C-C-A-U-Um-G-psi- G-C-U-m3C-U-G-C-A-C-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A- U-C-C-C-A-U-C-C-U-C-G-U-C-G-C-C-AOH. A comparison of the nucleotide sequence of tRNA(1Ser) from the bovine liver with already known sequences of serine tRNA revealed a number of common nucleotides, some of them, probably, participated in specific interaction with seryl-tRNA synthetase.     

Unusual anticodon loop structure found in E.coli lysine tRNA.

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.     

Substrate structural requirements of Schizosaccharomyces pombe RNase P

RNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent Km for two S. pombe tRNA precursors derived from the supS1 and sup3-e tRNA(Ser) genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.