HISTIDINE-TAGGED RNA-POLYMERASE - DISSECTION OF THE TRANSCRIPTION CYCLE USING IMMOBILIZED ENZYME

A stretch of six histidine residues (His6) has been genetically fused to the C terminus of the beta' polypeptide of Escherichia coli RNA pol ymerase. The His6-tagged beta' subunit assembles into RNA polymerase m olecules which perform all vital in vivo functions and behave qualitat ively normally in vitro. The HiS6 tag permits rapid purification of th e enzyme directly from crude cell extracts or from an in vitro reconst itution reaction by adsorption to Ni2+-chelating agarose resin, follow ed by elution with imidazole. The enzyme bound to the matrix remains t ranscriptionally active. The immobilized enzyme can withstand repeated buffer changes without substantial activity loss and permits controll ed stepwise 'walking' of the transcriptional complex along the DNA tem plate, and isolation of defined intermediates in the transcription cyc le. The immobilized RNA polymerase provides a powerful experimental sy stem for structural and functional analysis of RNA polymerase and its interaction with regulatory factors.