Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

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Herrera-Asmat O.
Lubkowska L.
Kashlev M.
Bustamante C.J.
Guerra D.G.
Kireeva M.L.
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Elsevier B.V.
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Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1–3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies.
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recombinant protein, bacterial protein, DNA directed RNA polymerase, holoenzyme, biosynthesis, chemistry, enzymology, Escherichia coli, genetic transcription