Relaxed control of RNA synthesis during nutritional shiftdowns of hisU mutant of Salmonella typhimurium.

Among the classes of histidine regulatory mutants isolated in Salmonella typhimurium, three of these mutants (hisT, hisW and hisU) exhibit pleiotrophic effects on the regulation of expression of other amino acid biosynthetic operons (1, 2). While the regulatory patterns of the hisT mutants are explained by the defective tRNA pseudouridylate synthetase (3), the exact function of the hisW and hisU loci are not as clearly defined, although both mutants exhibit reductions in the relative amino acid acceptance activity of several tRNA's (4). In studies of tRNA synthesis and processing in one such hisU mutant (hisU1820), we unexpectedly observed continued RNA synthesis during nutritional (carbon and energy source) transitions. It was also shown that this relaxed control of stable RNA formation is independent of the relA gene product.     

tRNA2Thr complements temperature sensitivity caused by null mutations in the htrB gene in Escherichia coli

According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr. In order to investigate the reason that this nonessential tRNA nevertheless exists in Escherichia coli, we attempted to isolate tRNA2Thr-requiring mutants. Using strain JM101F(-), which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr. These mutants had a mutation in the htrB gene, whose product is an enzyme involved in lipid A biosynthesis. Although it is known that some null mutations in the htrB gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity. We discuss a possible mechanism for the requirement for tRNA2Thr.     

Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase

Protein-RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure-function relationships involved in the interaction of the Escherichia coli tRNA(Asp) acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA(Asp) complex. Mutants were derived from Saccharomyces cerevisiae tRNA(Asp), which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA(Asp) knockout strain of E. coli. The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA(Asp) gene. The mutants provide a rich source of tRNA(Asp) sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.     

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.     

Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects

We investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) tRNA(UUR)(Leu). The studied substitutions are equivalent to the A3243G and T3250C human pathogenetic tRNA mutations. Our data show that both mutations can produce tRNA(UUR)(Leu) acylation defects, although to a different extent. For mutant A14G (equivalent to MELAS A3243G base substitution), the presence of the tRNA and its defective aminoacylation could be observed only in the nuclear context of W303, a strain where the protein synthesis defects caused by tRNA base substitutions are far less severe than in previously studied strains. For mutant T20C (equivalent to the MM/CPEO human T3250C mutation), the acylation defect was less severe, and a thermosensitive acylation could be detected also in the MCC123 strain. The correlation between the severity of the in vivo phenotypes of yeast tRNA mutants and those obtained in in vitro studies of human tRNA mutants supports the view that yeast is a suitable model to study the cellular and molecular effects of tRNA mutations involved in human pathologies. Furthermore, the yeast model offers the possibility of modulating the severity of yeast respiratory phenotypes by studying the tRNA mutants in different nuclear contexts. The nucleotides at positions 14 and 20 are both highly conserved in yeast and human mt tRNAs; however, the different effect of their mutations can be explained by structure analyses and quantum mechanics calculations that can shed light on the molecular mechanisms responsible for the experimentally determined defects of the mutants.     

A gene involved in modifying transfer RNA is required for fungal pathogenicity and stress tolerance of Colletotrichum lagenarium

7-Methylguanosine (m7G) modification of tRNA occurs widely in prokaryotes and eukaryotes, although information about its biological roles is limited. Here, we report that a gene involved in m7G modification of tRNA is required for infection by the phytopathogenic fungus Colletotrichum lagenarium. Analysis of the infection-deficient mutant of C. lagenarium, produced by plasmid insertional mutagenesis, identified a tagged gene that is designated APH1. The aph1 mutants, generated by targeted gene disruption, exhibit significant reduction in pathogenicity on the host plants. We conclude that APH1 is required for fungal infection in C. lagenarium. Aph1 showed a strong similarity to Saccharomyces cerevisiae Trm8 involved in m7G modification of tRNA. The m7G content of tRNA from the aph1 deletion mutant was severely reduced compared with that from the wild type, indicating that APH1 is required for m7G methyltransferase activity. Appressoria formed by the aph1 mutants developed penetration hyphae into cellophane, suggesting that appressoria of the mutants retain basic function for penetration. However, the aph1 mutants failed to develop intracellular penetration hyphae into epidermis of the host plants, suggesting a specific requirement of APH1 for appressorium-mediated host invasion. The mutants also had increased sensitivity to salinity and H2O2 stresses. Interestingly, a heat shock treatment on the host plants enabled the aph1 mutant to penetrate them. These data suggest that the APH1 is required for the plant invasion, probably to overcome environmental stresses derived from basal preinvasion (penetration) defence of the host plants.     

tRNA production links nutrient conditions to the onset of sexual differentiation through the TORC1 pathway

Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient‐rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.     

Isolation and Partial Characterization of Escherichia coli Mutants with Low Levels of Transfer Ribonucleic Acid Nucleotidyltransferase

To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5'-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3' terminal adenosine 5'-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5'-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.     

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.     

Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast

We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4

Summary Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models. It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA. Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation. The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study. It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA. The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.     

Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.     

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.     

Accumulation of pre-tRNA splicing ''2/3'' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.