- Title
- A vector system that allows simple generation of mutant Escherichia coli RNA polymerase
- Creator
- Yang, Xiao; Ma, Cong; Lewis, Peter
- Relation
- NHMRC.1008014
- Relation
- Plasmid Vol. 75, p. 37-41
- Publisher Link
- http://dx.doi.org/10.1016/j.plasmid.2014.06.002
- Publisher
- Academic Press
- Resource Type
- journal article
- Date
- 2014
- Description
- We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C-terminus of the b0 subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the b and b0 subunits (rpoB and rpoC, respectively), facilitating mutation of functionally significant subunit fragments through insertion of modified PCR fragments into the appropriate vector. RNAP with an R275A substitution in the b0 subunit, which is essential for interaction with transcription initiation factor r, was generated and exhibited reduced activity compared to native recombinant RNAP.
- Subject
- RNA polymerase; Escherichia coli; dual-expression; transcription
- Identifier
- http://hdl.handle.net/1959.13/1304578
- Identifier
- uon:20873
- Identifier
- ISSN:0147-619X
- Language
- eng
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