Regulation of transcription by translational components in coupled translation-transcription cell-free system
| Autor: | K.T. Schmeidler-Sapiro, T.R. Johnson, J. Ilan |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Male
Transcription Genetic RNA polymerase II Biology Biochemistry Ribosome RNA Transfer RNA Polymerase I Transcription (biology) Polysome RNA polymerase I Animals RNA Messenger Cell Nucleus Genetics Messenger RNA Cell-Free System RNA General Medicine Cell biology Liver RNA Ribosomal Protein Biosynthesis Transfer RNA biology.protein RNA Polymerase II Rabbits Chickens |
| Zdroj: | Biochimie. 74:495-510 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/0300-9084(92)90091-r |
| Popis: | A coupled translation-transcription cell-free system was established from eukaryotic cells. The biosynthetic activity of this coupled system closely resembles the synthetic behavior of cells in vivo , and exhibits regulatory phenomena similar to that of intact cells. The translational system consists of rabbit reticulocyte lysate, or its components fractioned by centrifugation. The transcriptional portion consists of cockerel liver nuclei. Incorporation of amino acids into protein by the coupled system is linear for hours. Similarly, transcription in the coupled system is continuous for hours and is proportional with time. More than 90% of the transcriptional products are secreted into the incubation medium. The components of the translational system influence and regulate transcriptional activities. In the presence of ribosomes the nuclei transcribe mostly poly(A) + RNA with α-amanitin sensitivity consistent with activation of RNA polymerase II. Hybrid selection experiments demonstrate authentic preproalbumin mRNA among the transcriptional products. The putative mRNA secreted into the medium in the coupled system is found on polysomes, indicating translation of de novo synthesized message. Addition of excess reticulocyte mRNP to the medium of the coupled system results in transcription of primarily ribosomal RNA, 5S RNA, and tRNA, the products of RNA polymerase I and III. These activities closely imitate the behavior of liver in vivo under conditions of nutritional shifts or hormonal influences. The coupled system transcribes, processes, and transports substantial quantities of RNA, about 1.6 μg/10 6 nuclei/h. Thus, a coupled system has been established that lends itself to the exploration of regulatory interactions of cell components as it appears to closely resemble the in vivo situation. |
| Databáze: | OpenAIRE |
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