Nucleoli formation under inhibited RNA synthesis

Formation and fusion of nucleoli after mitosis were studied in cultures of Chinese hamster cells and meristematic cells of Allium cepa and A. fistulosum under blocked RNA synthesis. To identify precisely which cells had passed through mitosis under actinomycin D blockade, cell cultures with micronuclei (induced by colchicine treatment), and binuclear Allium cells (induced by caffeine treatment), were used. It was found that in cells which have passed through mitosis after inhibition of RNA synthesis, the formation and fusion of nucleoli proceed more slowly than in cells not treated with actinomycin D; however, nucleoli appear and coalesce. Thus, telophase reconstruction of the nucleolus does not require simultaneous RNA synthesis and occurs at the expense of RNA that has been synthesized prior to mitosis.     

Control of ribosomal RNA synthesis by hematopoietic transcription factors

1. Download : Download high-res image (178KB)
2. Download : Download full-size image

     

Control of ribosomal RNA synthesis in Escherichia coli. III. Cytoplasmic factors for ribosomal RNA synthesis.

The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the "4S" fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation or rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acid-starved rel+ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.     

Stimulation of in vitro RNA synthesis by ribosomes and ribosomal proteins

RNA synthesis by purified RNA polymerase on bacteriophage T4 DNA in vitro was stimulated two- to threefold when 70 s ribosomes from Escherichia coli were present. This activity was retained in the ribosomes washed with NH₄Cl and thus free of translational initiation factors. Both ribosomal subunits, 30 s and 50 s, stimulated RNA synthesis. Stimulation by 50 s subunits ruled out the possibility that the stimulation of RNA synthesis is due to the ribosomes removing nascent RNA from the RNA-transcribing complex by specifically binding to the RNA. Instead, we found that the stimulation of RNA synthesis by ribosomes is related to the initiation process of RNA synthesis by RNA polymerase. Ribosomes stimulated the initial rate of RNA synthesis but had no effect on the rate of RNA chain elongation or the size of RNA produced. Stimulation of RNA synthesis by ribosomes required the presence of sigma factor. Delayed addition of ribosomes to the already initiated RNA-synthesizing system diminished the stimulatory effect of ribosomes. The stimulatory activity of ribosomes was recovered in the split protein fraction but not in the core material when 70 s ribosomes, 30 s and 50 s ribosomal subunits were banded in a CsCl equilibrium gradient. The activity was found only in acidic ribosomal proteins but not in basic ribosomal proteins. A few stimulatory acidic proteins were fractionated from both 30 s and 50 s ribosomal subunits by DE52 cellulose chromatography. The mode of stimulation of RNA synthesis by active ribosomal protein was similar to that by ribosomes and appears to be related to the initiation of RNA synthesis. Involvement of ribosomes or ribosomal proteins in the control of RNA synthesis through a positive control mechanism in conjunction with sigma factor is suggested.     

Increased messenger RNA from protein synthesis inhibited human fibroblasts

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal ³H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.     

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.