Kinetics of misincorporation and proofreading by bacterial RNA polymerase

Artificial transcription elongation compexes are assembled in vitro using synthetic deoxy-oligonucleotides (representing template and non template DNA strands), radiolabelled RNA (representing nascent transcript) and purified RNA polymerase. After high salt wash the incorrect NTP is added and reaction is allowed to proceed for the various amounts of time. Reaction is stopped by addition of formamide-containing loading solution and the products are resolved on high-percentage denaturing polyacryamide gel. Bands are revealed by PhosphoImaging (GE Healthcare) and analysed using ImageQuant software (GE Healthcare).

SEEK ID: https://fairdomhub.org/assays/131

Experimental Assay

Nikolay Zenkin

Projects: Noisy-Strep

Investigation: Wetlab approach to transcription fidelity

Study: Investigation of bacterial transcription fidelity and processivity

Assay type: Reactomics

Technology type: Enzymatic Activity Measurements

Organisms: No organisms

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Created: 20th Feb 2011 at 20:08

Last updated: 8th Nov 2017 at 15:21

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