Characterization of ribonucleic acid polymerase-T7 promoter binary complexes

Abstract

Several experimental approaches have been employed to independently determine rates of dissociation for Escherichia coli RNA polymerase from the T7 bacteriophage A1, A2, A3, and D promoters. Heparin challenge measurements employing abortive initiation turnover rates as an index of promoter occupancy were carried out over a range of heparin concentrations in order to separate intrinsic dissociation rates from dissociation due to direct heparin attack of the polymerase-promoter complex. Dissociation rates were found to vary widely even among major promoters considered to have equivalent strength in vivo. Direct heparin attack was found to occur slowly with respect to intrinsic dissociation of polymerase from most promoters. Results were verified by gel analysis of full-length transcripts after heparin challenge and by measuring abortive initiation rates after poly[d(A-T)] . poly[d(A-T)] challenge. In the latter case, it was discovered that the equilibrium distribution of polymerase between a promoter and poly[d(A-T)] . poly[d(A-T)] could be measured.