Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.

delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA.

Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity.

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.

Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA.

Analysis of the in vivo amber suppressor activity of mutants derived from two Escherichia coli serine tRNAs shows that substitution of 2 base pairs in the acceptor helix changes a serine suppressor tRNA to an efficient glutamine acceptor. Determination of the amino acid inserted in vivo into protein by this tRNA shows that these changes reduce the tRNA recognition by seryl-tRNA synthetase while increasing that of glutaminyl-tRNA synthetase. This implies that misaminoacylation in vivo is dependent on the competition by different synthetases for the tRNA. In addition, the "translational efficiency" of tRNA is an integral part in observing misaminoacylation in vivo.

Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors.

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.

A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya.

Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene products have been shown to function in making all of the pseudouridines found in tRNA and ribosomal RNA except for tRNA(Glu) pseudouridine13. In this work, a protein able to make this pseudouridine was purified by standard biochemical procedures. Amino-terminal sequencing of the isolated protein identified the synthase as YgbO. Deletion of the ygbO gene caused the loss of tRNA(Glu) pseudouridine13 and plasmid-borne restoration of the structural gene restored pseudouridine13. Reaction of the overexpressed gene product, renamed TruD, with a tRNA(Glu) transcript made in vitro also yielded only pseudouridine13. A search of the database detected 58 homologs of TruD spanning all three phylogenetic domains, including ancient organisms. Thus, we have identified a new wide-spread class of pseudouridine synthase with no sequence homology to the previously known four subgroups. The only completely conserved sequence motif in all 59 organisms that contained aspartate was GXKD, in motif II. This aspartate was essential for in vitro activity.