Open AccessThe Reconstitution of Escherichia coli DNA‐Dependent RNA Polymerase from Its Isolated Subunits
Author(s) -
PALM Peter,
HEIL Alfred,
BOYD Dana,
GRAMPP Bernd,
ZILLIG Wolfram
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb04067.x
Subject(s) - rna polymerase , enzyme , polymerase , dna , escherichia coli , kinetics , chemistry , protein subunit , microbiology and biotechnology , limiting , rna , yield (engineering) , biochemistry , biology , gene , mechanical engineering , physics , materials science , quantum mechanics , engineering , metallurgy
An improved method for the reconstitution of DNA‐dependent RNA polymerase from its isolated subunits in the absence of urea is described. It allows the kinetic analysis of the assembly mechanism. The rate of reconstitution as well as the final yield are strongly temperature‐dependent, whereas concentration has only a small effect. The kinetics of the formation of an intermediary complex α 2 β and an inactive enzyme complex α 2 ββ′σ were followed. The rate‐limiting and temperature‐sensitive step during reconstitution is the conversion of the inactive complex α 2 ββ′σ into active enzyme.