New England Biolabs, M0550S, E. coli RNA Polymerase, Core Enzyme

RNA Polymerase, Core Enzyme consists of 5 subunits designated α, α, β´, β, and ω. Related Categories RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Specification Unit Definition One unit is defined as the amount of enzyme required to incorporate 1 nmol NTP into RNA in 10 minutes at 37°C. Reaction Conditions 1X E.coli RNA Polymerase Reaction Buffer Incubate at 37°C 1X E.coli RNA Polymerase Reaction Buffer 40 mM Tris-HCl 150 mM KCl 10 mM MgCl2 1 mM DTT 0.01% Triton® X-100 (pH 7.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 100 mM NaCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.5 @ 25°C Unit Assay Conditions 1X E. coli RNA Polymerase Reaction Buffer, supplemented with 0.5 mM of each NTP, sigma factor 70 and 1 μg T7 phage DNA in 50 μl. FAQ Q: What is the difference between the E.coli RNA Polymerase, Core Enzyme and Holoenzyme? A: E. coli RNA Polymerase Core Enzyme consists of 5 subunits designated α, α, β', β, and ω. The enzyme is free of sigma factor and does not initiate specific transcription from bacterial and phage DNA promoters. The enzyme remains the ability to transcribe RNA from nonspecific initiation sequences. Addition of sigma factors will allow the enzyme to initiate RNA synthesis from specific bacterial and phage promoters. The core enzyme has a molecular weight of approximately 400 kDa. E. coli RNA Polymerase Holoenzyme is the core enzyme saturated with sigma factor 70. The Holoenzyme initiates RNA synthesis from sigma 70 specific bacterial and phage promoters. Q: What are the major functions of these subunits? A: The major functions of the subunits are: The β' subunit contains part of the active center responsible for RNA synthesis. The β subunit contains the rest of the active center responsible for RNA synthesis. The α subunit is present in two copies per molecule of RNA Polymerase. The α subunit contains determinants for assembly of RNA Polymerase and for interactions with regulatory factors. The small ω subunit facilitates assembly of RNA Polymerase and stabilizes assembled RNA Polymerase. Sigma factors, upon associating with the core enzyme, increase specificity for its promoters and allow transcription to initiate at correct sites. Q: Can the Core and Holoenzyme be used in PURExpress? A: Yes. If your gene has a strong sigma 70 promoter it can be translated in PURExpress by adding the Holoenzyme to the reaction. If your gene is under the control of other sigma factors you should use the core enzyme and the specific sigma factor. NEB has successfully used sigma factor 54 and the core enzyme in PURExpress. Q: What are the other sigma factors in E.coli? A: Besides the “housekeeping” sigma factor 70, other sigma factors in E.coli include: σ19 (FecI) - the ferric citrate sigma factor, regulates the fec gene for iron transport. σ24 (RpoE) - the extracytoplasmic/extreme heat stress sigma factor. σ28 (RpoF) - the flagellar sigma factor. σ32 (RpoH) - the heat shock sigma factor, it is turned on when exposed to heat. σ38 (RpoS) - the starvation/stationary phase sigma factor. σ54 (RpoN) - the nitrogen-limitation sigma factor.