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Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA
Abstract:
Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.
Insights
Researchers found that most RNA polymerase II activity in mouse cells is DNA-bound and concentrated at the nuclear skeleton. This suggests initiated RNA polymerase II molecules are non-randomly distributed along DNA loops.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear matrices serve as structural scaffolds within the cell nucleus.
- RNA polymerase II is the primary enzyme responsible for transcribing protein-coding genes.
- Understanding the localization and activity of RNA polymerase II is crucial for gene regulation studies.
Purpose of the Study:
- To investigate the distribution and activity of RNA polymerase II within isolated nuclear matrices.
- To determine if RNA polymerase II activity is associated with DNA in nuclear matrices.
- To explore the relationship between DNA content and RNA polymerase II activity in nuclear matrices.
Main Methods:
- Isolation of nuclear matrices from mouse plasmocytoma cells (P3-X63-Ag8.653).
- Quantification of RNA polymerase II activity in isolated nuclear matrices and original nuclei.
- Analysis of RNA polymerase II activity in relation to varying DNA content within nuclear matrices.
Main Results:
- Approximately 25% of total nuclear RNA polymerase II activity was recovered in isolated nuclear matrices.
- Only DNA-bound RNA polymerase II was detected in nuclear matrices, unlike in original nuclei which contained both free and DNA-bound forms.
- RNA polymerase II activity in nuclear matrices remained consistent across a wide range of DNA content (91% to 1.5%).
Conclusions:
- Initiated RNA polymerase II molecules are non-randomly distributed within the nucleus.
- RNA polymerase II activity is concentrated at DNA attachment points to the nuclear skeleton.
- Nuclear matrices play a significant role in organizing and anchoring active transcription machinery.