Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.     

Role of glutamine aminotransferase in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions

The involvement of glutamine aminotransferase activity in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions was studied. We were able to show that there are at least two different glutamine aminotransferase activities that are differentiated genetically, by their substrate specificity (pyruvate and glyoxylate dependence), and their different modes of regulation. The pyruvate-dependent glutamine aminotransferase activity plays a major role in glutamine catabolism under microaerophilic conditions since the wild-type strain S288C showed a 10-fold higher activity in static cultures than in agitated ones. The same strain also had 3-fold higher glutaminase B activity in agitated cultures than in static ones. Pyruvate-dependent glutamine aminotransferase activity is not regulated directly by O2 itself since a rho- strain showed a high activity regardless of the extent of aeration of cultures. Finally, we were able to isolate a mutant, strain CN20, derived from the rho- strain and unable to utilize glutamine as the sole nitrogen source, which was severely affected in pyruvate-dependent but not in glyoxylate-dependent aminotransferase activity.     

Identification of stop codon readthrough genes in Saccharomyces cerevisiae

We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI⁺] and [psi^–] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.     

Codon recognition during frameshift suppression in Saccharomyces cerevisiae.

A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene. Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified. By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing. Nonetheless, position 4 interactions influence the efficiency of suppression. Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides.     

Effects of tRNA-intron structure on cleavage of precursor tRNAs by RNase P from Saccharomyces cerevisiae.

RNase P derived from S. cerevisiae nuclei was tested for its ability to cleave a variety of naturally occurring and selectively altered precursor-tRNA molecules to yield matured 5' termini. Precursors were synthesized in vitro in order to test which aspects of substrate structure are crucial to recognition and cleavage by RNase P. Base modifications in the precursor substrates are not required for cleavage by the enzyme, but deletion and substitution mutations affecting any portion of the precursor tertiary structure reduce cleavage. In particular, a number of alterations in the intervening sequence (IVS) reduce the susceptibility of the substrate to cleavage by RNase P. The significance of these results is discussed in reference to the contribution of the IVS to the structure of the precursor-tRNA.     

Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae.

Production of glutamine synthetase in Saccharomyces cerevisiae is controlled by three regulatory systems. One system responds to glutamine levels and depends on the positively acting GLN3 product. This system mediates derepression of glutamine synthetase in response to pyrimidine limitation as well, but genetic evidence argues that this is an indirect effect of depletion of the glutamine pool. The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids. This system operates through the positive regulatory element GCN4. Expression of histidinol dehydrogenase, which is under general control, is not stimulated by glutamine limitation. A third system responds to purine limitation. No specific regulatory element has been identified, but depression of glutamine synthetase is observed during purine starvation in gln3 gcn4 double mutants. This demonstrates that a separate purine regulatory element must exist. Pulse-labeling and immunoprecipitation experiments indicate that all three systems control glutamine synthetase at the level of subunit synthesis.     

Purification and properties of glutamine synthetase from Saccharomyces cerevisiae

We have purified glutamine synthetase over 130-fold from Saccharomyces cerevisiae. The enzyme exhibits a Km for glutamate of 6.3 mM and a Km for ATP of 1.3 mM in the biosynthetic reaction, with a pH optimum from 6.1 to 7.0. Ten to twelve 43,000 molecular weight subunits comprise the active enzyme of 470,000 molecular weight. Rabbit antibodies prepared against the purified enzyme were used to show that induction of enzyme activity correlates with de novo synthesis of the enzyme subunit.     

Biochemical and physiological aspects of glutamine synthetase inactivation in Saccharomyces cerevisiae

Saccharomyces cerevisiae glutamine synthetase is inactivated in vivo by the addition of glutamine or ammonia. Inactivation is characterized by a specific loss of synthetase activity; transferase activity remains stable. Several physiological perturbations cause inactivation, such as carbon starvation or limitation for a required amino acid, which could cause a buildup of glutamine. The kinetics of reappearance of synthetase activity after inactivation suggest that the process is reversible in vivo. No change in the native size of the enzyme was associated with inactivation but there appears to be a change in the immunological properties of the enzyme subunit.     

Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.     

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.     

The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon.

We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon. Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified. In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2). In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus. The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300. The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence. sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process. Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction. As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4. Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta-galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction. The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon. This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene. However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA. In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2. The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon.     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

Unconventional codon reading by Mycoplasma mycoides tRNAs as revealed by partial sequence analysis.

Continuing our investigation of the tRNA genes and gene products in Mycoplasma mycoides, we report the sequence of the gene for tRNALeu (CAA) as well as partial primary structures of the following tRNAs: Leu (CAA), Leu (UAG), Arg (UCU), Thr (AGU) and Ile (CAU). It is suggested that in M. mycoides, at least some of the family codon boxes are read by only one tRNA each, using an unconventional method which does not discriminate between the nucleotides in the third codon position. M. mycoides is the first free-living organism known to use an unconventional method of this kind.     

ADP-binding to origin recognition complex of Saccharomyces cerevisiae

The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.     

Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases.

Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso-N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D-stem and/or in the anticodon stem.     

Cloning of a DNA sequence that complements glutamine auxotrophy in Saccharomyces cerevisiae

Glutamine (gln) requiring mutants of Saccharomyces cerevisiae have been isolated. They synthesize small amounts of glutamine synthetase (GS), which is more thermolabile than the enzyme from the parental strain. The gln auxotrophy was complemented in transformation experiments using an S. cerevisiae gene library constructed in the plasmid vector YEp13. The transformants were mitotically unstable and synthesized almost tenfold higher amounts of GS than wild-type cells. This activity was as thermoresistant as that from the wild-type strain. A recombinant plasmid was isolated from one of the transformants and partially mapped. Upon reintroduction into the auxotrophic strain, the transformation frequency to gln prototrophy was the same as that for the marker LEU2 gene. The evidence presented suggests that we have cloned the structural gene for GS from S. cerevisiae.     

A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall

To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.     

Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae.

Individual species of tRNA from Escherichia coli were treated with hydrazine/3 M NaCl to modify cytidine residues. The chemically modified tRNAs were used as substrate for ATP/CTP: tRNA nucleotidyltransferases from E. coli and yeast, with [alpha-32P]ATP as cosubstrate. tRNAs that were labeled were analyzed for their content of modified cytidines. Cytidines at positions 74 and 75 were found to be required chemically intact for interaction with both enzymes. C56 was also required intact by the E. coli enzyme in all tRNAs, and by the yeast enzyme in several instances. C61 was found to be important in seven of 14 tRNAs with the E. coli enzyme but only in four of 13 tRNAs with that from yeast. Our results support a model in which nucleotidyltransferase extends from the 3' end of its tRNA substrate across the top of the stacked array of bases in the accepter- and psi-stems to the corner of the molecule where the D- and psi-loops are juxtaposed.