Escherichia coli proteins eluted from mono Q chromatography, a final step during RNA polymerase purification procedure.

Publisher Summary This chapter reviews the preparation of highly pure and active RNA polymerase (RNAP) and discusses the essential step for biochemical and biophysical analysis of transcription. In Escherichia coli, the basic transcription machinery is the core RNAP, which consists of α 2 ββ. Although core RNAP is capable of elongation and termination at simple terminators, it requires the binding of a sigma factor to form a holoenzyme in order to be able to initiate transcription at promoters on DNA templates. The chapter discusses the major principle for the purification of RNAP is to take advantage of its ability to bind to DNA. As the core RNAP is in excess of σ 70 in the cell, in order to obtain a holoenzyme with a stoichimetric amount of σ 70 , it is important to separate core RNAP from the holoenzyme during preparations. This can be accomplished by the use of Mono Q chromatography with a shallow linear gradient.