2Chemical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3181; E-mail: firstname.lastname@example.org
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Received September 18, 2009; Revision received October 26, 2009
A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with σ70 subunit. Washing of the immobilized RNA polymerase with 5-10 mM solution of glutamate (pH 5.0-5.5) completely removed the σ70 subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between σ70 subunit and the core enzyme.
KEY WORDS: E. coli RNA polymerase, subunits, affinity chromatography, IMPACT system, transcription, amino acids