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  • Analysis of open complex formation during RNA polymerase II transcription initiation using heteroduplex templates and potassium permanganate probing.

Analysis of open complex formation during RNA polymerase II transcription initiation using heteroduplex templates and potassium permanganate probing.

Methods (San Diego, Calif.) (1997-07-01)
F C Holstege, H T Timmers
ABSTRACT

Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Potassium permanganate, ACS reagent, ≥99.0%, low in mercury
Sigma-Aldrich
Potassium permanganate, ≤150 μm particle size, 97%
Sigma-Aldrich
Potassium permanganate, ACS reagent, ≥99.0%
Sigma-Aldrich
Potassium permanganate solution, 2 mM
Sigma-Aldrich
Potassium permanganate solution, 0.1 M
Sigma-Aldrich
Potassium permanganate solution, 0.02 M
Sigma-Aldrich
Potassium permanganate solution, 0.01 M
Sigma-Aldrich
Potassium permanganate solution, 0.2 M
Sigma-Aldrich
Potassium permanganate solution, 5 mM