The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.     

Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.     

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild-type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase.     

Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.     

Terminal oxidation-reduction of yeast phenylalanine tRNA prevents donor and acceptor function at the peptidyl transferase center

Periodate oxidation of the ribose of the 3′-terminal adenosine of yeast tRNA^Phe followed by borohydride reduction has the net effect of splitting the C2′C3′ bond leaving two primary alcohol groups at these carbon atoms. This modified tRNA (tRNA^ox-red) could be acylated with phenylalanine but could not function as either a donor or acceptor at the peptidyl transferase center of the ribosome. Assays were performed with the phenylalanyl-pentanucleotides, CACCA^ox-red(acetylPhe) and CACCA^ox-red(Phe), which were isolated from the 3′-end of appropriately esterified tRNA^ox-red. Ado^ox-red(Phe) isolated from Phe-tRNA^ox-red was also inactive as an acceptor, but synthetic Ado^ox-red(Phe), a mixture of the 2′ and 3′ phenylalanyl esters, was active with an apparent K_m of 1.16 mM compared to 0.2 mM for control Ado(Phe). These results are interpreted to mean that (1) biosynthetic aminoacylation of tRNA^ox-red occurs specifically at the 2′-hydroxyl, (2) there is no 2′:3′ tautomerization in the ring-opened structure, and (3) peptidyl transferase recognizes specifically the 3′-aminoacyl esters of tRNA.     

Construction,aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon

The digestion of yeast initiator methionine tRNA with mung bean nuclease and U₂ ribonuclease yielded 5'- and 3'-fragments, respectively. These two fragments together represent the entire tRNA sequence except for A₃₅, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C₃₅ was reformed. Subsequent treatment of this product with CCA-transferase yielded a full-length methionine tRNA having an arginine CCU anticodon. This recombinant tRNA^Met (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNA^Met (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.     

The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA.

The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium. The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG). The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA.     

Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.     

Thio modification of yeast cytosolic tRNA is an iron-sulfur protein-dependent pathway

Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.     

tRNA import into yeast mitochondria is regulated by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, one of two cytosolic lysine-tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre-Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.     

Preparation of oligonucleotides corresponding to the acceptor stem of yeast tRNAPhe and their interaction with yeast ATP(CTP):tRNA nucleotidyltransferase

Seven oligonucleotides corresponding to the 3' and 5' sequences of the acceptor stem of yeast tRNAPhe have been prepared by chemical synthesis, chemical-enzymatic synthesis or by isolation from tRNA hydrolysates. The oligonucleotides have been examined as substrates for phosphodiester bond synthesis in the presence of ATP as catalysed by yeast ATP (CTP): tRNA nucleotidyltransferase. Oligonucleotides which correspond to the sequence of the 3'-strand of the tRNA acceptor stem and possess no secondary structure exhibit little or no activity with the enzyme. The ability of the enzyme to catalyse the synthesis of a phosphodiester linkage using ATP and an oligonucleotide corresponding to the 3'-strand of the acceptor stem is in general dramatically increased when an oligonucleotide corresponding to the sequence of the 5'-strand of tRNA acceptor stem is present. In cases where significant activity was observed kinetic parameters have been determined.