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T7 RNA polymerase cannot transcribe through a highly knotted DNA template
J Portugal1, A Rodríguez-Campos
1Departamento de Biología Molecular y Celular, Centro de Investigacióny Desarrollo, CSIC, Barcelona, Spain.
Nucleic Acids Research
|December 15, 1996
Summary
Highly knotted DNA, unlike other topological forms, is not a template for transcription by T7 RNA polymerase. Unknotting restores transcription capacity, suggesting DNA topology regulates gene expression.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA topology, including supercoiling and knotting, influences DNA metabolic processes.
- RNA polymerase activity is crucial for gene expression and can be affected by DNA structure.
Purpose of the Study:
- To investigate the impact of DNA topological forms, specifically knotting, on T7 RNA polymerase transcription in vitro.
- To determine if highly knotted DNA is a functional template for transcription.
Main Methods:
- In vitro transcription assays using T7 RNA polymerase.
- Analysis of transcription on linear, supercoiled, relaxed, and variably knotted plasmid DNA templates.
- Enzymatic manipulation of DNA topology using yeast topoisomerase II.
Main Results:
- T7 RNA polymerase transcribed linear, supercoiled, and relaxed DNA templates efficiently.
- Transcription levels varied with DNA knotting; highly knotted DNA fractions were refractory to transcription.
- Unknotting of DNA using catalytic topoisomerase II restored transcription to levels comparable to other topological forms.
Conclusions:
- Highly knotted DNA is the sole topological form that serves as a non-template for transcription.
- DNA topological state, particularly the degree of knotting, plays a regulatory role in transcription.
- The presence of DNA nodes may be a mechanism for regulating transcription.