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Analysis of Translation Initiation During Stress Conditions by Polysome Profiling
Published on: May 19, 2014
Translational regulation of polysome formation during dormancy of Physarum polycephalum
Abstract:
The translational activity of actively growing microplasmodia and dormant microsclerotia of Physarum polycephalum was investigated by analyzing the distribution of ribosomes in polysomes. Microplasmodial post-mitochondrial fractions contained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period which preceded microsclerotium formation, polysome levels remained constant, whereas the subunit titer began to increase. During encystment ribosomal subunits continued to accumulate as the level of polysomes gradually decreased. Dormant microsclerotia contained a large surplus of stored ribosomal subunits but no detectable polysomes. However, incubation of microsclerotia with concentrations of cycloheximide sufficient to slow polypeptide elongation without affecting initiation caused the gradual reappearance of polysomes at the expense of the subunits. Under these conditions the percentage of subunits driven into polysomes reached values similar to those of actively growing microplasmodia. Microsclerotia returned to nutrient medium contained very low levels of polysomes during the lag period which preceded germination. These were formed with preexisting, stored messenger ribonucleic acid. During the germination period, polysome levels were markedly increased. This elevation was dependent on new ribonucleic acid transcription. It is concluded that dormant microsclerotia contain functional messenger ribonucleic acid and ribosomes which are subject to translational repression at the level of initiation.
Insights
Dormant Physarum polycephalum microsclerotia store ribosomes and functional messenger RNA. Translational repression at the initiation level explains their dormancy, with protein synthesis resuming upon germination.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Physarum polycephalum exhibits distinct life stages: actively growing microplasmodia and dormant microsclerotia.
- Understanding the regulation of protein synthesis is crucial for comprehending cellular transitions between growth and dormancy.
Purpose of the Study:
- To investigate the translational activity of Physarum polycephalum during different life stages.
- To elucidate the mechanisms regulating protein synthesis in dormant microsclerotia.
Main Methods:
- Analysis of ribosome distribution in polysomes from microplasmodial and microsclerotial fractions.
- Treatment of microsclerotia with cycloheximide to assess translational control.
- Monitoring polysome levels and ribosomal subunit accumulation during starvation, encystment, and germination.
Main Results:
- Actively growing microplasmodia showed high polysome levels and few ribosomal subunits.
- Dormant microsclerotia contained abundant stored ribosomal subunits but lacked detectable polysomes.
- Cycloheximide treatment induced polysome formation in microsclerotia, indicating stored functional messenger RNA.
- Germination involved a lag phase with low polysomes, followed by increased polysome levels dependent on new RNA transcription.
Conclusions:
- Dormant Physarum polycephalum microsclerotia possess functional messenger RNA and ribosomes.
- Translational repression, specifically at the initiation level, maintains microsclerotial dormancy.
- Reactivation involves the utilization of stored components and de novo transcription for protein synthesis.
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