Pseudouridine Modifications Research Articles

Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes.

In Saccharomyces cerevisiae, pseudouridine formation in the middle position of the tRNA(Tyr) anticodon (psi 35) is dependent on the presence of the intron in the tRNA(Tyr) gene (Johnson and Abelson, Nature 302:681-687, 1983). Drosophila melanogaster tRNA(Tyr) genes contain introns of three size classes: 20 or 21 base pairs (bp) (six genes), 48 bp (one gene), and 113 bp (one gene). As in yeast, removal of the intron led to loss of psi 35 in the anticodon when transcription was assayed in Xenopus laevis oocytes. All Drosophila intron sizes supported psi 35 formation. The same results were obtained with the homologous X. laevis tRNA(Tyr) genes containing introns of 12 or 13 bp or with a deleted intron. The introns of yeast (Nishikura and DeRobertis, J. Mol. Biol. 145:405-420, 1981), D. melanogaster, and X. laevis tRNA(Tyr) wild-type genes, while they all supported psi 35 synthesis, did not share any consensus sequences. As discussed, these results, taken together, suggest that for appropriate function the psi 35 enzyme in the X. laevis oocyte needs the presence of an unqualified intron in the tRNA gene and a tRNA(Tyr)-like structure in the unprocessed tRNA precursor.

Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes.

In Saccharomyces cerevisiae, pseudouridine formation in the middle position of the tRNA(Tyr) anticodon (psi 35) is dependent on the presence of the intron in the tRNA(Tyr) gene (Johnson and Abelson, Nature 302:681-687, 1983). Drosophila melanogaster tRNA(Tyr) genes contain introns of three size classes: 20 or 21 base pairs (bp) (six genes), 48 bp (one gene), and 113 bp (one gene). As in yeast, removal of the intron led to loss of psi 35 in the anticodon when transcription was assayed in Xenopus laevis oocytes. All Drosophila intron sizes supported psi 35 formation. The same results were obtained with the homologous X. laevis tRNA(Tyr) genes containing introns of 12 or 13 bp or with a deleted intron. The introns of yeast (Nishikura and DeRobertis, J. Mol. Biol. 145:405-420, 1981), D. melanogaster, and X. laevis tRNA(Tyr) wild-type genes, while they all supported psi 35 synthesis, did not share any consensus sequences. As discussed, these results, taken together, suggest that for appropriate function the psi 35 enzyme in the X. laevis oocyte needs the presence of an unqualified intron in the tRNA gene and a tRNA(Tyr)-like structure in the unprocessed tRNA precursor.

An unusual genetic link between vitamin B6 biosynthesis and tRNA pseudouridine modification in Escherichia coli K-12.

We characterized several unusual phenotypes caused by stable insertion mutations in a gene that is located upstream in the same operon from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. Mutants containing kanamycin resistance (Kmr) cassettes in this upstream gene, which we temporarily designated usg-2, failed to grow on minimal plus glucose medium at 37 and 42 degrees C. However, usg-2::Kmr mutants did form oddly translucent, mucoid colonies at 30 degrees C or below. Microscopic examination revealed that cells from these translucent colonies were spherical and seemed to divide equatorially. Addition of D-alanine restored the shape of the mutant cells to rods and allowed the mutants to grow slowly at 37 degrees C and above. By contrast, addition of the common L-amino acids prevented growth of the usg-2::Kmr mutants, even at 30 degrees C. Furthermore, prolonged incubation of usg-2::Kmr mutants at 37 and 42 degrees C led to the appearance of several classes of temperature-resistant pseudorevertants. Other compounds also supported growth of usg-2::Kmr mutants at 37 and 42 degrees C, including glycolaldehyde and the B6 vitamers pyridoxine and pyridoxal. This observation suggested that usg-2 was pdxB, which had been mapped near hisT. Complementation experiments confirmed that usg-2 is indeed pdxB, and inspection of the pyridoxine biosynthetic pathway suggests explanations for the unusual phenotypes of pdxB::Kmr mutants. Finally, Southern hybridization experiments showed that pdxB and hisT are closely associated in several enterobacterial species. We consider reasons for grouping pdxB and hisT together in the same complex operon and speculate that these two genes play roles in the global regulation of amino acid metabolism.

Isolation and characterization of antisuppressor mutations in Escherichia coli.

Nonsense mutations in lacI have been shown to be useful as indicators of the efficiency of nonsense suppression. From strains containing supE and a lacI nonsense mutation, selection for LacI- mutants has resulted in the isolation of four antisuppressor mutations. Tn10 insertions linked to these mutations were isolated and used to group the four mutations into three loci. The asuA1 and asuA2 mutations are linked to trp, reduce suppression by supE approximately twofold, and affect a variety of suppressors. The asuB3 mutation was mapped by P1 cotransduction to rpsL but does not confer resistance to streptomycin. The asuC4 mutation reduced suppression by supE by 95% and was shown biochemically to result in the loss of two pseudouridine modifications from the 3' side of the anticodon stem and loop of tRNA2Gln. This mutation is linked to purF, suggesting that it is a new allele of hisT.

The yeast tRNATyr gene intron is essential for correct modification of its tRNA product.

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.

Biosynthesis of Pseudouridine in Transfer Ribonucleic Acid

An enzyme that catalyzes the formation of specific pseudouridine residues in the anticodon region of tRNA has been characterized in extracts of Salmonella typhimurium and Escherichia coli. It has previously been shown that hisT regulatory mutants of S. typhimurium lack the pseudouridine modifications in the anticodon region of histidine and several other tRNA species. This has permitted the development of a simple radiochemical assay for the modification enzyme, based on the release of tritium from hisT tRNA isolated from cells grown in [5-3H]uridine. This enzyme activity is absent from hisT mutants and is relatively heat-labile in extracts from temperature-sensitive hisT mutants, which suggests that the modification enzyme itself is the primary gene product of the hisT gene.

Mutant tRNA His ineffective in repression and lacking two pseudouridine modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons1–4. Histidyl-tRNAHis has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNAHis as the wild type4 and in the one example sequenced, contains tRNAHIS with a structure identical to that of the wild type8. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNAHISin vitro2. The amounts of charged and uncharged tRNAHis present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined9. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNAHisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNAHis. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNAHis) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNAHis which these mutants contain.