RNA polymerase B from Drosophila melanogaster larvae. Purification and partial characterization

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA-dependent RNA polymerase B (or II) is a crucial enzyme in eukaryotic transcription.
• Understanding its properties is essential for deciphering gene expression regulation.

Purpose of the Study:

• To purify DNA-dependent RNA polymerase B from Drosophila melanogaster.
• To characterize its enzymic and structural properties.
• To investigate its relationship with homologous enzymes from other eukaryotes.

Main Methods:

• Purification of RNA polymerase B from third-instar larvae of Drosophila melanogaster.
• Enzymic assays to determine substrate preference and sensitivity to alpha-amanitin.
• Dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for polypeptide analysis.
• Immunological characterization using an antiserum against Drosophila RNA polymerase B.

Main Results:

• Essentially pure DNA-dependent RNA polymerase B was obtained from Drosophila.
• The enzyme exhibits properties similar to eukaryotic RNA polymerases, including preferential transcription of single-stranded DNA and inhibition by alpha-amanitin.
• SDS-PAGE revealed ten distinct polypeptides, with four major components (polypeptides 1-4) present in constant molar ratios.
• An antiserum against Drosophila RNA polymerase B inhibited homologous and heterologous RNA polymerase B activity in vitro, but only precipitated the Drosophila enzyme.

Conclusions:

• The purified Drosophila RNA polymerase B is a complex enzyme with conserved eukaryotic features.
• The study provides insights into the subunit composition and immunological properties of Drosophila RNA polymerase B.
• The findings contribute to the comparative understanding of RNA polymerase II across different species.