Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.     

Molecular events during the release of delta-aminolevulinate dehydratase from catabolite repression.

Transfer of exponential-phase cells of Saccharomyces cerevisiae, previously grown in 2% glucose, to a derepression medium resulted in a prompt increase in the level of delta-aminolevulinate dehydratase, the rate-limiting enzyme of heme biosynthesis under these conditions. This derepression exhibited a lag of 35 min at 23 degrees C and required the participation of both RNA and protein syntheses. Dissection of the molecular events during this lag period disclosed that RNA synthesis, rnal gene function (messenger RNA transport from nucleus to cytosol), and initiation of protein synthesis were completed within less than 10, 18, and 24 min, respectively. The potential regulation of derepression by mitochondrial gene products and mitochondrial function was probed by means of a series of isogenic, respiration-deficient (rho-, pet-, and mit-) mutants; no such regulation was found.     

Lomofungin inhibition of allophanate hydrolase synthesis in Saccharomyces cerevisiae

Summary The RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and -glucosidase) as well as that for gross protein synthesis. In both cases the studies conclude that cognate messenger RNAs decay with a half life of approximately 20 minutes. This antibiotic has been used to determine the half life of allophanate hydrolase specific synthetic capacity. We find that it decays with a half life of about three minutes; a value that agrees with the decay rates of allophanate hydrolase synthetic capacity following removal of inducer. These observations argue that mRNA may be metabolized by two separate routes in Saccharomyces.     

Molecular events associated with induction of arginase in Saccharomyces cerevisiae.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.     

Stable Messenger Ribonucleic Acid and Germination of Myxococcus xanthus Microcysts

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.     

Metabolite compartmentation in Saccharomyces cerevisiae.

Uninduced cultures of Saccharomyces cerevisiae exhibit high basal levels of allantoinase, allantoicase, and ureidoglycolate hydrolase, the enzymes responsible for degrading allantoin to urea. As a result, these activities increase only 4- to 8-fold upon induction, whereas the urea-degrading enzymes, urea carboxylase and allophanate hydrolase, have very low basal levels and routinely increase 30-fold on induction. Differences in the inducibility of these five enzymes were somewhat surprising because they are all part of the same pathway and have the same inducer, allophanate. Our current studies reconcile these observations. S. cerevisiae normally contained up to 1 mM allantoin sequestered in a cellular organelle, most likely the vacuole. Separation of the large amounts of allantoin and the enzymes that degrade it provide the cell with an efficient nitrogen reserve. On starvation, sequestered allantoin likely becomes accessible to these degradative enzymes. Because they are already present at high levels, the fact that their inducer is considerably removed from the input allantoin is of little consequence. This suggests that at times metabolite compartmentation may play an equal role with enzyme induction in the regulation of allantoin metabolism. Metabolism of arginine, another sequestered metabolite, must be controlled both by induction of arginase and compartmentation because arginine serves both as a reserve nitrogen source and a precursor of protein synthesis. The latter function precludes the existence of high basal levels of arginase.     

Macromolecular synthesis, differentiation and cell division in Tetrahymena pyriformis, mating type I variety 1

In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of ¹⁴C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of ¹⁴C-uracil into cold-TCA-insoluble material, after a 5–10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis. Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10–15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.     

Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.     

Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

The synthesis of ribonucleic acid (RNA) and of protein in Escherichia coli during glucose-lactose diauxie lag have been examined. The rate of RNA synthesis is about 7%, of the corresponding rate during exponential growth and the rate of protein synthesis 10 to 15%. Inhibition of RNA synthesis occurs to the same extent in both rel and rel(+) strains. The RNA which accumulates during 20 min in diauxie lag is composed of about 50% ribosomal and transfer RNA species and about 50% of a fraction which resembles messenger RNA (mRNA) in its heterogeneous sedimentation properties. Decay of the heterogeneous fraction occurs in the presence of glucose and actinomycin D with a half-life of 3 min, the same as that of pulse-labeled mRNA; however, during the diauxie lag, the half-life of this RNA is about 25 min. Accumulation of the heterogeneous RNA is further increased when protein synthesis is blocked by chloramphenicol. The data suggest that the disproportionate accumulation of mRNA during diauxie lag and energy source shift-down may be attributed at least in part to increased stability of mRNA, but do not rule out a preferential synthesis of mRNA.     

Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

$\text{Saccharomyces cerevisiae}$ can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, “CO₂”, and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes.     

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism

Growth substrates containing an s-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid. Over a 25-year period, a number of original research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. In the present study, allophanate, not urea, was shown to be the 2-nitrogen intermediate in cyanuric acid metabolism in all the bacteria examined. Six different experimental results supported this conclusion: (i) synthetic allophanate was shown to readily decarboxylate to form urea under acidic extraction and chromatography conditions used in previous studies; (ii) alkaline extraction methods were used to stabilize and detect allophanate in bacteria actively metabolizing cyanuric acid; (iii) the kinetic course of allophanate formation and disappearance was consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments; (iv) protein extracts from cells grown on cyanuric acid contained allophanate hydrolase activity; (v) genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were found in several cyanuric acid-metabolizing bacteria; and (vi) TrzF, an AtzF homolog found in Enterobacter cloacae strain 99, was cloned, expressed in Escherichia coli, and shown to have allophanate hydrolase activity. In addition, we have observed that there are a large number of genes homologous to atzF and trzF distributed in phylogenetically distinct bacteria. In total, the data indicate that s-triazine metabolism in a broad class of bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease.     

A suppressor of temperature-sensitive rna mutations that affect mRNA metabolism in Saccharomyces cerevisiae.

We have isolated a dominant suppressor of rna mutation (SRN1) that relieves the temperature-sensitive inhibition of mRNA synthesis of ribosomal protein genes in the yeast Saccharomyces cerevisiae. The suppressor was selected for its ability to alleviate simultaneously the temperature-sensitive growth phenotypes of rna2 and rna6. Several independently isolated suppressors appeared to be recessive lethal mutations. One suppressor, SRN1, was recovered as viable in haploid strains. SRN1 can suppress rna2, rna3, rna4, rna5, rna6, and rna8 singly or in pairs, although some combinations of rna mutations are less well suppressed than others. The suppressor allows strains with rna mutations to grow at 34 degrees C but is unable to suppress at 37 degrees C; however, SRN1 does not, by itself, prevent growth at 37 degrees C. In addition, SRN1 suppresses the rna1 mutation which affects general mRNA levels and also leads to the accumulation of precursor tRNA for those tRNAs that have intervening sequences. SRN1 can suppress the rna1 mutation as well as the rna1 rna2 double mutation at 34 degrees C. The suppressor does not affect the temperature-sensitive growth of two unrelated temperature-sensitive mutations, cdc4 and cdc7.     

Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae.