Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.     

The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.     

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.     

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.     

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.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.     

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.