Purification of Core Enzyme of <i>Escherichia coli </i>RNA Polymerase by Affinity Chromatography
A method for isolation of E. coli RNA-polymerase core enzyme based on the use of IMPACT technology and determination of the effect of dissociation of а-subunit from the holoenzyme under the action of low concentrations of free amino acids were developed. Supplementary treatment of immobilized RNA polymerase with 5-10 mM solution of glutamate at pH 5-5.5 allowed quantitative elution of а-subunit and most of the associated protein impurities from the column. The possibility to reconstruct the RNA polymerase holoenzyme directly on the affinity column was demonstrated. The functional activity of resulting enzyme preparations was demonstrated in experiments on in vitro transcription. The influence of some individual amino acids and their mixtures on various stages of transcription initiation was investigated. The results show the specific destruction of σ-core enzyme interactions in the presence of amino acids.