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

• Autores: Herrera-Asmat O., Lubkowska L., Kashlev M., Bustamante C.J., Guerra D.G., Kireeva M.L.
• Formato: artículo
• Fecha de Publicación: 2017
• Institución: Consejo Nacional de Ciencia Tecnología e Innovación
• Repositorio: CONCYTEC-Institucional
• Lenguaje: inglés
• OAI Identifier: oai:repositorio.concytec.gob.pe:20.500.12390/751
• Enlace del recurso: https://hdl.handle.net/20.500.12390/751; https://doi.org/10.1016/j.pep.2017.03.013
• Nivel de acceso: acceso abierto
• Materia: recombinant protein; bacterial protein; DNA directed RNA polymerase; holoenzyme; biosynthesis; chemistry; enzymology; Escherichia coli; genetic transcription; https://purl.org/pe-repo/ocde/ford#3.00.00

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.