Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.
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. 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 α2ββ'ω·σAcan 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.
Jason Choy Laboratory of Single Molecule Biophysics, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Laboratorio de Moléculas Individuales, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martin de Porras, Lima-31, Peru.
Omar
O
Herrera-Asmat
NCI Center for Cancer Research, Frederick, MD 21702, USA.
Lucyna
L
Lubkowska
NCI Center for Cancer Research, Frederick, MD 21702, USA.
Mikhail
M
Kashlev
Jason Choy Laboratory of Single Molecule Biophysics, Department of Molecular and Cell Biology, Department of Physics and Department of Chemistry, Kavli Energy Nanoscience Institute, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. Electronic address: carlosb@berkeley.edu.
Carlos J
CJ
Bustamante
Laboratorio de Moléculas Individuales, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martin de Porras, Lima-31, Peru. Electronic address: daniel.guerra@upch.pe.
Daniel G
DG
Guerra
NCI Center for Cancer Research, Frederick, MD 21702, USA. Electronic address: kireevam@mail.nih.gov.
Maria L
ML
Kireeva
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D020035:Holoenzymes; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases
Elongation complex assembly; Open complex; Promoter initiation