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Specific role of manganese and magnesium on RNA synthesis in rabbit bone marrow erythroid cell nuclei
Abstract:
Specific roles of manganese (Mn) and magnesium (Mg) on the activities of DNA-dependent RNA polymerases I and II isolated from rabbit bone marrow erythroid cell nuclei were investigated. Three main polymerases were separated from the cell nuclei. When RNA polymerase I and Mg were added to the RNA synthesis assay mixture containing erythroid cell DNA as template, RNA transcription activity was highest, but when Mg was replaced with Mn, denatured calf thymus DNA formed a better template than erythroid cell DNA. In contrast, nucleoplasmic DNA from erythroid cell and liver DNA were the best templates to stimulate RNA transcription when RNA polymerase II and Mn were added to the assay mixture. However, if Mn was replaced with Mg, RNA synthesis activity was drastically reduced when the template was nucleoplasmic DNA of erythroid cell. RNA polymerase I and Mg synthesized GC rich RNA, whereas RNA polymerase II and Mn synthesized AU rich RNA. Sedimentation analysis showed that the molecular weights of the RNA produced by polymerase I were larger when the enzyme was activated with Mg than with Mn, whereas those of the RNA produced by polymerase II were larger with Mn than with Mg. Furthermore, RNA produced by polymerase I and Mg using chromatin as a template hybridized better with nucleolar DNA than with nucleoplasmic DNA, whereas that produced by polymerase II and Mn hybridized better with nucleoplasmic DNA than with nucleolar DNA. These results suggest that RNA synthesis is dependent on the activity of specific RNA polymerases and the presence of specific divalent cations and templates, and that the cofactor and template for RNA polymerase I are, respectively, Mg and the nucleolar DNA of cell nuclei, whereas those for RNA polymerase II are Mn and nucleoplasmic DNA.
Insights
Magnesium (Mg) activates RNA polymerase I for GC-rich RNA synthesis using nucleolar DNA, while manganese (Mn) activates RNA polymerase II for AU-rich RNA synthesis using nucleoplasmic DNA in rabbit bone marrow cells.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- DNA-dependent RNA polymerases are crucial for gene expression.
- Specific divalent cations and DNA templates influence polymerase activity.
Purpose of the Study:
- To investigate the distinct roles of manganese (Mn) and magnesium (Mg) in the activity of RNA polymerases I and II.
- To identify specific cofactors and DNA templates for each polymerase type.
Main Methods:
- Isolation and separation of RNA polymerases I and II from rabbit bone marrow erythroid cell nuclei.
- In vitro RNA synthesis assays using different divalent cations (Mg, Mn) and DNA templates (erythroid cell DNA, calf thymus DNA, nucleoplasmic DNA, liver DNA, chromatin).
- Sedimentation analysis to determine RNA molecular weights and hybridization studies to assess RNA-DNA interactions.
Main Results:
- RNA polymerase I activity was highest with Mg and erythroid cell DNA, producing GC-rich RNA with higher molecular weight that hybridized with nucleolar DNA.
- RNA polymerase II activity was optimal with Mn and nucleoplasmic or liver DNA, yielding AU-rich RNA with lower molecular weight that hybridized with nucleoplasmic DNA.
- Denatured calf thymus DNA served as a better template for polymerase I when Mg was replaced by Mn.
Conclusions:
- RNA synthesis is critically dependent on specific RNA polymerases, divalent cations, and DNA templates.
- RNA polymerase I utilizes Mg as a cofactor and nucleolar DNA as a template.
- RNA polymerase II employs Mn as a cofactor and nucleoplasmic DNA as a template.