Purification and Characterization of the DNA-Dependent RNA Polymerase and its Subunit σ fromMicrococcus luteus

Publisher Website
Google Scholar
Abstract
DNA-dependent RNA polymerase [EC 2.7.7.6] from M. luteus can be isolated from cell extracts after removal of an excess of nucleic acids by fractionation with (NH4)2SO4, followed by 2 consecutive gel filtrations through agarose and chromatography on cellulose phosphate. Either homogeneous holoenzyme or a mixture of core and holoenzyme is obtained in this way, as is indicated by electrophoresis in polyacrylamide gels in the absence of detergent, where core enzyme migrates ahead of holoenzyme. Homogeneous core enzyme can be isolated from holoenzyme by chromatography on DEAE-cellulose. Core enzyme contains the subunits .alpha., .beta. and .beta.'' previously described in a molar ratio of 2:1:1. Holoenzyme contains an additional subunit .sigma. of 80,000 MW (molar subunit composition .alpha.2 .beta..beta.''.sigma.) and 2 relatively small polypeptides (MW 14,000 and 25,000, respectively). Subunit .sigma. may be isolated from holoenzyme by chromatography on DEAE-cellulose at pH 6.9 in the presence of low concentrations of glycerol. The behavior of holoenzyme during sedimentation in a glycerol gradient at low ionic strength indicates its occurrence as a dimer of the .alpha.2 .beta..beta.''.sigma.-protomer, but the monomeric form is preferred by core enzyme. Holoenzyme is much more active than core enzyme in RNA synthesis on bacteriophage T4 DNA as template. The activity of the latter is stimulated by isolated .sigma.. M. luteus .sigma. and holoenzyme enhances also the activity of core enzyme from Eschierichia coli. The formation of a hybrid between micrococcal .sigma. and E. coli core polymerase is also suggested by the influence of .sigma. on the oligomerisation of the enzyme from E. coli.

This publication has 18 references indexed in Scilit: