We hope to produce a recombinant mycobacterial RNA polymerase through heterologous expression in E. coli to evaluate the activity of some drug candidates as RNAP inhibitors.
Unfortunately, up until now, we have only found references that propose methods for High Throughput Screening (HTS) and that is not our goal.
Does anyone know a method that could be applied in a smaller scale, preferably that does not include using radioactive reagents?
If you are going to use purified RNA polymerase, then you can measure the production of pyrophosphate that results from RNA polymerization using a plasmid and 4 ribonucleotides as the substrate. A colorimetric method for doing this assay with E. coli RNA polymerase is given in this paper:
Article Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Swi...
You should include a plasmid-free control because there could be some background nucleotide hydrolysis in the absence of the plasmid.
Adam B Shapiro
Thank you so much for your answer!
The paper and the method proposed surely will be of aid to us.
Best regards.
Hello Dr. João Vítor Perez de Souza
i suggest you these papers :-
1- DNA template sequence control of bacterial RNA polymerase escape from the promoter
https://academic.oup.com/nar/article/46/9/4469/4931255
2- Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Switch Region Inhibitors with Confirmed Mode of Action in Haemophilus influenzae
Article Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Swi...
3- Structural Biology of Bacterial RNA Polymerase
Article Structural Biology of Bacterial RNA Polymerase
4- Real-time measurement of in vitro transcription
Article Real-time measurement of in vitro transcription
5- New insights into the bacterial RNA polymerase inhibitor CBR703 as a starting point for optimization as an anti-infective agent.
https://hzi.openrepository.com/bitstream/handle/10033/326174/Zhu%20et%20al_final.pdf?sequence=1&isAllowed=y
Hello Dr. João Vítor Perez de Souza
i suggest you these papers :-
1- DNA template sequence control of bacterial RNA polymerase escape from the promoter
https://academic.oup.com/nar/article/46/9/4469/4931255
2- Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Switch Region Inhibitors with Confirmed Mode of Action in Haemophilus influenzae
Article Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Swi...
3- Structural Biology of Bacterial RNA Polymerase
Article Structural Biology of Bacterial RNA Polymerase
4- Real-time measurement of in vitro transcription
Article Real-time measurement of in vitro transcription
5- New insights into the bacterial RNA polymerase inhibitor CBR703 as a starting point for optimization as an anti-infective agent.
https://hzi.openrepository.com/bitstream/handle/10033/326174/Zhu%20et%20al_final.pdf?sequence=1&isAllowed=y
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