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Double-stranded RNA in chromatin transcripts formed by exogenous RNA polymerase
Summary
In vitro transcribed RNA contains double-stranded RNA (dsRNA) segments, resistant to RNase. These dsRNA structures form via intramolecular base pairing and are more prevalent in chromatin transcripts.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- In vitro transcription produces RNA molecules.
- RNA structure can influence its function and stability.
- Understanding RNA secondary structures is crucial for molecular biology.
Purpose of the Study:
- To investigate the presence and characteristics of RNase-resistant structures in in vitro transcribed RNA.
- To determine the nature of these structures and the factors influencing their formation.
- To compare RNA secondary structures transcribed from DNA versus chromatin templates.
Main Methods:
- In vitro transcription using rat liver and Escherichia coli RNA polymerases.
- RNase resistance assays with specific RNases for double-stranded or hybrid RNA.
- Denaturation kinetics analysis.
- G-C base pair content analysis.
Main Results:
- Transcribed RNA contains significant RNase-resistant, double-stranded RNA (dsRNA) sequences formed by intramolecular base pairing.
- These dsRNA segments range from 20-30 nucleotides, with some exceeding 100 nucleotides, and are G-C rich (60-65%).
- Chromatin transcripts exhibit higher proportions of dsRNA than DNA transcripts, and homologous RNA polymerase yields more dsRNA than bacterial polymerase.
Conclusions:
- In vitro transcription generates structured RNA with intramolecular dsRNA elements.
- Chromatin structure and homologous RNA polymerase favor dsRNA formation.
- Endogenous chromatin RNA polymerase, lacking initiation activity, does not produce dsRNA.
- Preliminary data suggests 5' ends of transcripts may be enriched in complementary sequences.