The regulation of RNA synthesis in yeast II: Amino acids shift-up experiments

Summary A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15–20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of the regulation of RNA synthesis are discussed.Paper I in this series is Oliver and McLaughlin (1977)     

The regulation of RNA synthesis in yeast IV

Summary A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.Paper III in this series is Elliott and McLaughlin (Molec. Gen. Genet., In press)     

Initiation of enzymatic DNA synthesis by yeast RNA polymerase I.

In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.     

Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.     

Insulin regulation of RNA synthesis

During periods of nitrogen exportation from the cell, mitochondrial carbamoyl phosphate is synthesized, thus initiating the urea cycle. During times of nitrogen conservation by the liver cell, carbamoyl phosphate is synthesized in the cytosol of the cell, whereupon the de novo pyrimidine synthesis pathway is initiated. The de novo pathway provides pyrimidines for increased ribonucleic acid synthesis. Formerly, it was believed that these two pathways functioned irrespective of one another. However, recent experimental evidence indicates that, when excess ammonia is present, mitochondrial carbamoyl phosphate passes from the mitochondria into the cell cytosol, where it is metabolized by the de novo pyrimidine synthesis pathway. When ornithine and excess ammonia are both present, mitochondrial carbamoyl phosphate no longer passes from the mitochondria into the cytosol to be metabolized by the de nova pathway. Thus the metabolic fate of mitochondrial carbamoyl phosphate, and that of excess nitrogen, is determined by the presence or absence of ornithine. In turn, this key molecule is the substrate for the cytoplasmic enzyme ornithine decarboxylase. When ornithine decarboxylase is stimulated by insulin, ornithine is metabolized to putrescine. The activated ornithine decarboxylase combines with ribonucleic acid polymerase, activating the later enzyme. When ornithine is acted upon by ornithine decarboxylase, it is no longer available for the perpetuation of the urea cycle and mitochondrial carbamoyl phosphate levels rise until the carbamoyl phosphate passes into the cytosol to be metabolized by the de novo pathway. Increased amounts of pyrimidines are available for the activated ribonucleic acid polymerase. Therefore insulin, through its stimulation of ornithine decarboxylase, achieves cellular nitrogen retention by regulating nitrogen incorporation into newly synthesized ribonucleic acid.     

Regulation of mitochondrial ribosomal RNA synthesis in yeast

Summary Studies were undertaken to determine if mitochondrial rRNA synthesis in yeast is regulated by general cellular stringent control mechanism. Those variables affecting the relaxation of a cycloheximide-induced stringent response as a result of medium-shift-down or tyrosine limitation include: 1) the stage of cell growth, 2) carbon source, 3) strain differences and, 4) integrity of the cell wall. The extent of phenotypic relaxation decreased or was eliminated entirely in a strain dependent manner as cells entered stationary phase of growth or by growth of cells on galactose or in osmotically stabilized spheroplast cultures.Cytoplasmic and mitochondrial RNA species were extracted from regrowing spheroplast cultures subjected to different experimental regimens and analyzed by electrophoresis on 2.5% polyacrylamide gels. Relative rates of synthesis were determined in pulse experiments and normalized by double-label procedures to longterm label material. Tyrosine starvation was found to inhibit synthesis of the large and small rRNA species of both cytoplasmic and mitochondrial rRNAs to about 5–20% of the control values. Chloramphenicol inhibits mitochondrial and cytoplasmic rRNA synthesis to 60–80% of control; however, chloramphenicol addition does not relax the stringent inhibition of either class of rRNAs. Cycloheximide addition results in 70–80% inhibition of synthesis of both cellular species of rRNAs. As noted above, cycloheximide does not relax the stringent response of cytoplasmic rRNA synthesis in spheroplasts, and also does not relax the stringent inhibition of mitochondrial rRNA synthesis. From these studies, we conclude that both cytoplasmic and mitochondrial rRNA synthesis share common control mechanisms related to regulation of protein synthesis by shift-down or amino acid limitation.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

The background of the total synthesis of yeast alanine transfer RNA

<正>The research findings concerning the total synthesis of yeast alanine transfer RNA (yeast alanine tRNA) were successively published in Chinese Science Bulletin (1982) and Science in     

Differential regulation of rat insulin I and II messenger RNA synthesis: effects of fasting and cyproheptadine

Rats and mice retain a duplicated insulin (I) gene. Because the duplicated gene shares only incomplete homology with the ancestral insulin (II) gene it may be regulated differently. In the studies presented here we measured changes in abundance of these distinct insulin mRNAs and their precursors in response to fasting and fasting plus a single dose of cyproheptadine, two experimental manipulations that cause changes in the level of total insulin mRNA in rats. Both diminished rat insulin II mRNA to a greater extent than rat insulin I mRNA. Rat insulin II mRNA comprised 41% of the total insulin mRNA in 0 h controls and decreased to 33% of the total insulin mRNA after a 10-h fast. Insulin II mRNA decreased to 26% of the total insulin mRNA 10 h after treatment with cyproheptadine. To determine whether these manipulations had effects on insulin mRNA synthesis, precursors for each of the two mRNAs were quantified. Fasting for 24 h had only small effects on insulin I mRNA precursor, but diminished rat insulin II pre-mRNA to 32% of the 0 h control values. One and a half hours after fasting plus cyproheptadine administration, pre-mRNA for rat insulin II levels had decreased to 38%, while rat insulin I pre-mRNA remained at levels present in 0 h controls. Levels of rat insulin I and II pre-mRNAs were both maximally depressed at 10 h, but rat insulin II pre-mRNA decreased to 3%, while rat insulin I pre-mRNA diminished to only 49% of controls.(ABSTRACT TRUNCATED AT 250 WORDS)