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Coli RNA Polymerase (coli + rna_polymerase)

Distribution by Scientific Domains
Chemistry75%

Kinds of Coli RNA Polymerase

  • escherichia coli rna polymerase
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    Selected Abstracts

    The interaction between ,S, the stationary phase , factor, and the core enzyme of Escherichia coli RNA polymerase

    GENES TO CELLS, Issue 3 2002
    Frédéric Colland
    Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure ,2,,,) associated with one of the , subunits, required for promoter recognition. Different , factors compete for core binding. Among the seven , factors present in E. coli, ,70 controls gene transcription during the exponential phase, whereas ,S regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled ,S and ,70, we compared the affinities of both , factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that ,S binds to the core enzyme with fivefold reduced affinity compared to ,70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of ,S, while increasing the accessibility in region 4.1 of ,S. We have also analysed the positioning of ,S on the holoenzyme by the proximity-dependent protein cleavage method using ,S derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the , and ,, subunits, but not ,. Regions 2.5, 3.1 and 3.2 of ,S are close to both , and ,, subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using ,S and previous data from ,70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2. [source]

    Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

    PROTEIN SCIENCE, Issue 6 2010
    Baley A. Fong
    Abstract A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP , subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the ,, ,,, and , subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo -assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target. [source]

    Structure of the Escherichia coli RNA polymerase , subunit C-terminal domain

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010
    Samuel Lara-González
    The , subunit C-terminal domain (,CTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli,CTD (, subunit residues 245,329) determined to 2.0,Ĺ resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P21 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, Rfree = 0.236) has improved geometry compared with prior lower resolution determinations of the ,CTD structure [Jeon et al. (1995), Science, 270, 1495,1497; Benoff et al. (2002), Science, 297, 1562,1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of ,CTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction. [source]