A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Mercurated nucleoside triphosphates offer a method for distinguishing newly synthesized RNA from endogenous RNA.
• Chicken oviduct chromatin serves as a model system for studying gene transcription.

Purpose of the Study:

• To investigate the DNA-dependent transcription of specific mRNA sequences, such as ovalbumin mRNA, using mercurated nucleoside triphosphates and E. coli RNA polymerase.
• To analyze the fidelity and origin of newly synthesized RNA during in vitro transcription of chromatin.

Main Methods:

• Transcription of chicken oviduct chromatin using E. coli RNA polymerase and mercurated nucleoside triphosphates.
• Purification of newly synthesized RNA using SH-agarose chromatography.
• Analysis of specific mRNA sequences (ovalbumin mRNA, globin mRNA) using hybridization techniques.
• Inhibition studies with actinomycin D.

Main Results:

• Apparent preferential production of ovalbumin mRNA sequences was observed, but this was not inhibited by actinomycin D, suggesting a non-DNA-dependent process.
• Globin mRNA, when added to the transcription assay, was retained on SH-agarose, indicating interaction with the system.
• Copurification of chromatin-associated RNA with synthesized mercurated RNA was primarily due to RNA-dependent synthesis of complementary sequences by the bacterial enzyme.
• Denaturation of transcripts reduced contamination with endogenous RNA but did not confirm DNA-dependent messenger RNA sequence synthesis.

Conclusions:

• The study questions the DNA-dependent synthesis of specific mRNA sequences under the described conditions.
• The observed RNA synthesis appears to be RNA-dependent, with the bacterial enzyme producing complementary RNA strands.
• Further investigation is needed to definitively demonstrate DNA-dependent transcription of specific messenger RNA sequences using this method.