A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.     

Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation

We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.     

Evolution of a mitochondrial tRNA PHE gene in A. thaliana: import of cytosolic tRNA PHE into mitochondria

Previously we have described a putative tRNATyr in Arabidopsis thaliana mitochondria, the sequence of which is different from that of other plant mitochondrial tRNATyr genes. We show here that this tRNATyr gene sequence is present in several copies in the mitochondrial genome of A. thaliana. One copy of these tRNATyr gene sequences, termed here tRNATyr-1, could encode a functional tRNA. Expression analysis has shown that the tRNATyr-1 gene is cotranscribed with the downstream tRNAGlu gene, and that the corresponding mature-sized tRNA is present in mitochondria. We also show that the native tRNATyr gene, similar to the mitochondrial tRNATyr genes found in plants, is present in the A. thaliana mitochondrial genome and expressed. The tRNATyr-1 gene has been previously suggested to be derived from a tRNAPhe gene sequence. We show here that, as a consequence, there is no tRNAPhe gene in the mitochondrial genome of A. thaliana and that a cytosolic tRNAPhe is imported in A. thaliana mitochondria.     

The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of approximately 70% in the tRNASer(UCN) level and a decrease of approximately 45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to approximately 40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located approximately 7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.     

The role of exportin-t in selective nuclear export of mature tRNAs

Exportin-t (Xpo-t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP-loaded Ran. Xpo-t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells. We examine the mechanism by which Xpo-t-RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors. Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TPsiC and acceptor arms of tRNAPhe and Xpo-t-RanGTP. Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5' and 3' end-processing of tRNA affects Xpo-t-RanGTP interaction and nuclear export, while aminoacylation is not essential. Intron-containing, end-processed, pre-tRNAs can be bound by Xpo-t-RanGTP and are rapidly exported from the nucleus if Xpo-t is present in excess. These results suggest that at least two mechanisms are involved in discrimination of pre-tRNAs and mature tRNAs prior to nuclear export.     

In vivo efficacy of antimicrobial-coated fabric from prosthetic heart valve sewing rings

Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3'-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3'-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3'-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis. Valyl-tRNA(Val) terminating in 3'-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3' end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3' cytosine. Similar observations were made in studies of the interaction of 3' end mutants of E. coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3' end of aminoacyl-tRNAs.     

Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.     

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.     

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).