Inhibition of protein synthesis by an efficiently expressed mutation in the yeast 5.8S ribosomal RNA
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Abstract:
Recent studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides strongly suggested that this RNA plays an important role in eukaryotic ribosome function. To evaluate this possibility further, a ribosomal DNA transcription unit from Schizosaccharomyces pombe was cloned into yeast shuttle vectors with copy numbers ranging from 2 to approximately 90 per cell; to allow direct detection of expressed RNA and to disrupt the function of the 5.8S rRNA molecule, a five base insertion was made in a universally conserved GAAC sequence. The altered mobility of the mutant RNA was readily detected by gel electrophoresls and analyses indicated that mutant RNA transcription reflected the ratio of plasmid to endogenous rDNA. The highest copy number plasmid resulted in about 40 – 50% mutant RNA. This mutant RNA was readily integrated into the ribosome structure resulting in an in vivo ribosome population which was also about 40 – 50% mutant; the rates of growth and protein synthesis were equally reduced by approximately 40%. A comparable level of inhibition in protein synthesis was demonstrated in vitro and polyribosomal profiles revealed a consistent increase in size. Subsequent RNA analyses indicated a normal distribution of mutant RNA in both monoribosomes and polyribosomes, but elevated tRNA levels in mutant polyribosomes. Additional mutations in alternate GAAC sequences revealed similar but cumulative effects on both protein synthesis and polyribosome profiles. Taken together, these results suggest little or no effect on initiation but provide in vivo evidence of a functional role for the 5.8S rRNA in protein elongation.Keywords:
5.8S ribosomal RNA
Polysome
Transcription
Human genetics
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RNA species from the haploid gametophyte generation of the moss Tortula ruralis exhibit typical eukaryotic characteristics. The major ribosomal and soluble RNA species are stable during drying and rehydration. RNA synthesis occurs rapidly on reintroduction of the moss to water and incorporation into high molecular weight RNA fractions was detected after 20 to 30 minutes of rehydration and into low molecular weight fractions after 30-60 minutes. Newly synthesized ribosomal RNA was detected in ribosomes within 2 hours of rehydration, but not in polysomes. It is apparent that the ribosomal and transfer RNA conserved during desiccation is involved in the re-establishment of early protein synthesis during subsequent rehydration and that, initially, there is no requirement for newly synthesized material.
Polysome
Desiccation Tolerance
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The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.
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Polysome
5.8S ribosomal RNA
Eukaryotic Ribosome
Ribosomal protein
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In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated.
Polysome
Eukaryotic Ribosome
23S ribosomal RNA
Ribosomal protein
5.8S ribosomal RNA
50S
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5.8S ribosomal RNA
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Rat liver polysomes, single ribosomes, EDTA‐produced subparticles and free ribosomal RNA were treated with formaldehyde and their properties were studied. The following results were obtained. RNA can be isolated from fixed polysomes or subparticles by exhaustive digestion with pronase. Such RNA shows a normal electrophoretic behaviour in agar gel. In free RNA treated with formaldehyde the mobility of 18‐S and 28‐S RNA fractions is reduced; latent breaks are not revealed by heating; sensitivity towards ribonuclease is increased. No such effects are observed with RNA treated with formaldehyde while in the particles. In fixed polysomes or single ribosomes the two subparticles appear to be covalently bound through their proteins. No intermolecular bonding occurs between 18‐S and 28‐S RNA either in fixed polysomes or in free RNA. The fixed ribosomes and subparticles can be fractionated by agar gel electrophoresis and give profiles similar to those of untreated particles. These results may prove useful in combining CsCl density gradient fractionation with gel electrophoretic methods.
Polysome
Pronase
Pancreatic ribonuclease
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Agarose
Eukaryotic Ribosome
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The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.
Polysome
Ribosomal protein
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