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
Projects: Noisy-Strep
Investigation: Wetlab approach to transcription fidelity
Study: Investigation of bacterial transcription fidelity and processivity
Assay position:
Assay type: Reactomics
Technology type: Enzymatic Activity Measurements
Organisms: No organisms
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Created: 20th Feb 2011 at 19:08
Last updated: 8th Nov 2017 at 14:21
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Projects: Noisy-Strep
Institutions: University of Newcastle
SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".
The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.
Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...
Projects: BaCell-SysMO, COSMIC, SUMO, KOSMOBAC, SysMO-LAB, PSYSMO, SCaRAB, MOSES, TRANSLUCENT, STREAM, SulfoSys, SysMO DB, SysMO Funders, SilicoTryp, Noisy-Strep
Web page: http://sysmo.net/
Bistable switches are the key elements of the regulatory networks governing cell development, differentiation and life-strategy decisions. Transcriptional noise is a main determinant that causes switching between different states in bistable systems. By using the human pathogen Streptococcus pneumoniae as a model bacterium, we will investigate how transcriptional fidelity and processivity influence (noisy) gene expression and participate in bistability. To study this question, we will use both ...
Programme: SysMO
Public web page: http://www.sysmo.net/index.php?index=163
Handling and manipulation of S. pneumoniae using molecular, cell biological and genetic tools.
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Investigation: Wetlab approach to transcription fidelity
Assays: Kinetics of misincorporation and proofreading by bacterial RNA polymerase
