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

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dc.contributor.author Herrera-Asmat, Omar
dc.contributor.author Lubkowska, Lucyna
dc.contributor.author Kashlev, Mikhail
dc.contributor.author Bustamante, Carlos J.
dc.contributor.author Guerra, Daniel G.
dc.contributor.author Kireeva, Maria L.
dc.date.accessioned 2019-01-25T16:03:18Z
dc.date.available 2019-01-25T16:03:18Z
dc.date.issued 2017
dc.identifier.uri https://hdl.handle.net/20.500.12866/4737
dc.description.abstract 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 omega 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. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than alpha2betabeta'omega.sigma(A) can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD. en_US
dc.language.iso eng
dc.publisher Elsevier
dc.relation.ispartof urn:issn:1096-0279
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject Bacterial Proteins/biosynthesis/chemistry/genetics/isolation & purification en_US
dc.subject DNA-Directed RNA Polymerases/biosynthesis/chemistry/genetics/isolation & purification en_US
dc.subject Promoter Regions, Genetic en_US
dc.subject Transcription, Genetic en_US
dc.subject Elongation complex assembly en_US
dc.subject Escherichia coli/genetics/metabolism en_US
dc.subject Holoenzymes/biosynthesis/chemistry/genetics/isolation & purification en_US
dc.subject Mycobacterium tuberculosis/enzymology/genetics en_US
dc.subject Open complex en_US
dc.subject Promoter initiation en_US
dc.subject Recombinant Proteins/biosynthesis/chemistry/genetics/isolation & purification en_US
dc.title Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.1016/j.pep.2017.03.013
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.02.00 es_PE
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.04.00


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