We wish to inactivate RNAP in E. coli, specifically the transcription elongation phase, and preferably without disassembly of the transcription elongation complex. The well-known rifampicin is inhibiting the initiation step allowing run-out of the existing transcriptions. We used previously streptolydigin that inhibits the elongation, it worked well with B. subtilis but does not penetrate the E. coli envelope (and is anyway not manufactured presently). Can anybody recommend another antibiotic with the required properties or a suitable temperature-sensitive RNAP mutant?
Dear Itzhak,
Do you wish to inactivate RNAP in vitro or in vivo?
If in vitro, it is possilbe to "walk" RNAP to the desired position by adding a subset of NTPs followed by washing of the complex (to remove the unincorporated NTPs), and then adding another subset of NTPs. An even better possibility is to simply assemble the elongation complex on a nucleic acid scaffold of your choice - we did this to study disassembly of elongation complexes and it works excelently - the complex is active - when NTPs are added, RNAP starts transcribing. When factors of choice are added, you can observe how they affect the elongation complex disassembly.
See
Article The torpedo effect in Bacillus subtilis : RN ase J1 resolves...
Dear Libor,
Thank you for suggesting an elegant solution in vitro. The problem is that we wish to do this in vivo.
Hi Itzhak, check the Microcin J25; it should inhibit transcription by binding within and obstructing the RNAP secondary channel - acting essentially as a "cork in a bottle", preventing NTPs to get in the active centre. But I am not sure about penetration to different organisms.
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