Comparative studies of the effect of DNA superhelicity on in vitro transcription catalyzed by Escherichia coli S100 proteins and purified RNA polymerase.

Abstract

The effect of DNA superhelicity on in vitro transcription catalyzed by purified Escherichia coli RNA polymerase or S100 crude extract proteins was examined at various KCl concentrations. DNA from a recombinant plasmid pMT48 harboring the pL promotor-controlled fused N-trp genes and the pR promotor-controlled tof (cro) gene was employed as a template. Stimulation of transcription by superhelicity is generally more pronounced with the S100 crude extract proteins than with pure RNA polymerase. At KCl concentrations lower than 100 mM with pure RNA polymerase, there is no significant difference in the template activity between the supercoiled and relaxed forms of pMT48 DNA. In contrast, the dependence of efficient template activity on superhelicity is great over a whole range of KCl concentrations from 1.7 to 400 mM in the system using the S100 crude extract. The relative insensitivity of the pR promotor to superhelicity can be observed in either transcription assay system. Analysis of the kinetics of pL-promoted synthesis of trp mRNA indicates that diminished transcription in vitro on a relaxed template results mainly from less frequent RNA chain initiations, but at least in part from premature arrest of the chain elongation.