Protocol for E. coli RNA Polymerase Holoenzyme

E. coli RNA Polymerase Holoenzyme
Cat. Nos. S90050 and S90250
E. coli RNA Polymerase Holoenzyme plays a central role in controlling gene expression
in the bacterium through the synthesis of RNA transcripts. Epicentre’s preparation of
Holoenzyme is 100% saturated with sigma subunit (σ70) and thus initiates RNA synthesis
specifically at promoter sequences on native bacterial or bacteriophage DNA.* A method
to assay enzyme function is presented in Chamberlain et al. 1
The enzyme is supplied at a unit concentration of 1 U/μl (0.50 μg/μl, 2.0 x 10 3 U/mg) and
is purified from the rifampicin-sensitive strain BL21.
Product Specifications
Storage: Store only at –70°C.
Storage Buffer: E. coli RNA Polymerase Holoenzyme is supplied in a 50% glycerol
solution containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1 mM EDTA, and 1 mM
dithiothreitol (DTT).
Unit Definition: One unit catalyzes the incorporation of 1 nmol of ribonucleoside
triphosphates (NTPs) into RNA in 10 minutes at 37°C.
Quality Control: E. coli RNA Polymerase Holoenzyme is function-tested in a reaction
containing: 1X E. coli RNA Polymerase Transcription Buffer, 2 mM DTT, 0.25 mM NTPs, and
1 μg T7 D111 DNA as template. The enzyme activity is assayed in a reaction containing:
1X E. coli RNA Polymerase Transcription Buffer, 10 mM DTT, 0.5 mM NTPs, and 1 μg T7
D111 DNA as template.
5X E. coli RNA Polymerase Transcription Buffer: 0.2 M Tris-HCl (pH 7.5), 0.75 M KCl,
50 mM MgCl 2
, and 0.05% Triton® X-100. Optimum buffer composition, including the
concentration of DTT, will vary based on the experimental application.
Contaminating Activity Assays: E. coli RNA Polymerase Holoenzyme is free of
detectable DNA exo- and endonuclease, and RNase activities.
Reference:
1. Chamberlin, M.J. et al., (1979) J. Biol. Chem. 254, 10061.
*Holoenzyme refers to E. coli RNA Polymerase with the sigma 70 (σ 70 ) as determined by SDS-PAGE analysis and by initiation of transcription
from the host promoter on bacteriophage T7 DNA. The presence of other minor sigma factors (e.g., σ 35 ), transcription factors (e.g., GreA or
GreB), or transcription accessory proteins are not excluded.
www.epicentre.com Lit. # 014 • 6/2011 1

E. coli RNA Polymerase Holoenzyme
Related Products: The following products are also available:
– E. coli RNA Polymerase Core Enzyme
– NTP Solutions
– Tagetin RNA Polymerase Inhibitor
– E. coli DNA Polymerase I
Tagetin is a trademark of Epicentre, Madison, Wisconsin.
Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.
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