Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120

Abstract

A procedure for the purification of RNA polymerase from vegetative cells of the filamentous cyanobacterium Anabaena 7120 is described. Polyethyleneimine precipitation followed by gel filtration and affinity chromatography steps results in greater than 99% purification with 46% yield. The enzyme has a novel core component of Mr = 66,000, designated gamma, in addition to the typical prokaryotic beta'beta alpha 2 core enzyme. The sigma subunit has been identified by reconstitution of specific transcriptional activity from core enzyme and gel-purified sigma. In transcription assays, this RNA polymerase initiates at a number of Anabaena vegetative cell promoters, as well as from a bacteriophage T4 early promoter, but does not initiate at nitrogen fixation (nif) promoters used in heterocysts. The promoter specificity of Anabaena RNA polymerase is compared with that of Escherichia coli RNA polymerase.