Control of nitrogen catabolite repression is not affected by the tRNAGln-CUU mutation, which results in constitutive pseudohyphal growth of Saccharomyces cerevisiae.
Saccharomyces cerevisiae responds to nitrogen availability in several ways. (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR). Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell's doubling time. (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth. (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G1. A specific allele of the SUP70/CDC65 tRNAGln gene (sup70-65) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR. Our data confirm that pseudohyphal growth occurs gratuitously in sup70-65 mutants cultured in nitrogen-rich medium at 30 degrees C. However, we find neither any defect in NCR in the sup70-65 mutant nor any alteration in the control of YVH1 expression, which has been previously shown to be specifically induced by nitrogen starvation. (+info)
The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl-tRNA synthetase from Escherichia coli.
We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) and showed that the set is sufficient to switch the identity of tRNAGln to Glu [Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996) J. Mol. Biol. 256, 685-700]. In the present study, we attempted to switch the identity of tRNAAsp, which has a sequence similar to that of tRNAGlu, and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1*C72, and U11*A24). A simple transplantation of the rest of the major determinants (U34, U2*A71, U13*G22**Alpha46, and Delta47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu. To confer an optimal glutamate accepting activity to tRNAAsp, two other elements, C4*G69 in the middle of the acceptor stem and C12*G23**C9 in the augmented D helix, were required. Consistently, the two base pairs, C4*G69 and C12*G23, in tRNAGlu had been shown to exist in the interface with glutamyl-tRNA synthetase (GluRS) by phosphate-group footprinting. We also found the two elements in the framework of tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln to Glu in the previous study. By the identity-determinant set (C4*G69 and C12*G23**C9 in addition to U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved. (+info)
Magnesium-dependent alternative foldings of active and inactive Escherichia coli tRNA(Glu) revealed by chemical probing.
A stable conformer of Escherichia coli tRNA(Glu), obtained in the absence of Mg(2+), is inactive in the aminoacylation reaction. Probing it with diethylpyrocarbonate, dimethyl sulfate and ribonuclease V1 revealed that it has a hairpin structure with two internal loops; the helical segments at both extremities have the same structure as the acceptor stem and the anticodon arm of the native conformer of tRNA(Glu)and the middle helix is formed of nucleotides from the D-loop (G15-C20:2) and parts of the T-loop and stem (G51-C56), with G19 bulging out. This model is consistent with other known properties of this inactive conformer, including its capacity to dimerize. Therefore, this tRNA requires magnesium to acquire a conformation that can be aminoacylated, as others require a post-transcriptional modification to reach this active conformation. (+info)
Genealogy of families of SINEs in cetaceans and artiodactyls: the presence of a huge superfamily of tRNA(Glu)-derived families of SINEs.
Several novel (sub)families of SINEs were isolated from the genomes of cetaceans and artiodactyls, and their sequences were determined. From comparisons of diagnostic nucleotides among the short interspersed repetitive elements (SINEs) in these (sub)families, we were able to draw the following conclusions. (1) After the divergence of the suborder Tylopoda (camels), the CHRS family of SINEs was newly created from tRNA(Glu) in a common ancestor of the lineages of the Suina (pigs and peccaries), Ruminantia (cows and deer), and Cetacea (whales and dolphins). (2) After divergence of the Suina lineage, the CHR-1 SINE and the CHR-2 SINE were generated successively in a common ancestor of ruminants, hippopotamuses, and cetaceans. (3) In the Ruminantia lineage, the Bov-tA SINE was generated by recombination between the CHR-2 SINE and Bov-A. (4) In the Suina lineage, the CHRS-S SINE was generated from the CHRS SINE. (5) In this latter lineage, the PRE-1 family of SINEs was created by insertion of part of the gene for tRNA(Arg) into the 5' region of the CHRS-S family. The distribution of a particular family of SINEs among species of artiodactyls and cetaceans confirmed the most recent conclusion for paraphyly of the order Artiodactyla. The present study also revealed that a newly created tRNA(Glu)-derived family of SINEs was subjected both to recombination with different units and to duplication of an internal sequence within a SINE unit to generate, during evolution, a huge superfamily of tRNA(Glu)-related families of SINEs that are now found in the genomes of artiodactyls and cetaceans. (+info)
A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.
The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation. (+info)
Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase. Predominant and distinct roles of the mnm5 and s2 modifications of U34.
Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP. (+info)
Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana.
The complete nucleotide sequence of the mitochondrial genome of the Oriental white stork, Ciconia boyciana, has been determined from captive storks by a novel method incorporating Long PCR and shotgun sequencing. 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes were identified as in other vertebrate mitochondrial genomes. The position and direction of the NADH6 and tRNA-Glu genes were the same as previously reported for avian mitochondrial genomes. A 71 bp direct repeat and long CAAA repeat sequences were found at the 3' end of the D-loop region, together with SCB-1, SCB-2, SCB-3, and three TAS sequences. Direct sequencing of the PCR fragments in the D-loop region in 26 captive Oriental white storks originating from Japan, China, and Russia revealed nucleotide differences at 18 sites along 1,248 bp, and a total of nine haplotypes have been identified. It was found that one pair of individuals in the Japanese captive breeding program were of the same haplotype, suggesting that they were caught from the same nest. The pair has since been dissolved in consideration of the possibility of inbreeding depression. (+info)
Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs.
Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell. (+info)