WRS-85D: A Tryptophanyl-tRNA Synthetase Expressed to High Levels in the Developing Drosophila Salivary Gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase

Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNAIle) at its active site. The misactivated amino acid is then translocated to an editing site located >25 Å away. The role of the misactivated amino acid in determining the rate of translocation is not known. Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, α-aminobutyrate, and cysteine. In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, α-aminobutyrate, and cysteine. Although their rates of misactivation are clearly distinct, their rates of translocation are similar. Thus, the rate of translocation is independent of the nature of the misactivated amino acid. This result suggests that the misactivated amino acid per se has little or no role in directing translocation.

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TΨC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Δ cells lacked m1A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m1A)methyltransferase [(m1A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m1A in either total tRNA or purified tRNAiMet lacking only this modification. Kinetic analysis of the purified complex revealed KM values for AdoMet or tRNAiMet of 5.0 μM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m1A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression

The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i6A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i6A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i6A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.

Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph

Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these “cells within cells” lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)7AAG6A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.

Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase: Role of a 16-amino acid insertion module in initiator tRNA recognition

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC 2.1.2.9) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles. The determinants for formylation in the tRNA are clustered mostly in the acceptor stem. As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in Escherichia coli MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem. We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources. A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not. The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation. These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.

PLMItRNA, a database for mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

The PLMItRNA database for mitochondrial tRNA molecules and genes in Viridiplantae (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159–162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (Rhodophytae) and two Stramenopiles. The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA.

Aminoacyl-tRNA synthetases database

Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases’ evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and Söll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202–236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobase s.ibch.poznan.pl/aars/.

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS–suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The “21st synthetase–tRNA pairs” include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules1

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.