Studies of the binding of Escherichia coli RNA polymerase to DNA : III. Tight binding of RNA polymerase holoenzyme to single-strand breaks in T7 DNA

Abstract

Single-strand breaks in T7 DNA enhance transcription by Escherichia coli core RNA polymerase, probably by providing new sites for RNA chain initiation. Significant enhancement is obtained with a small number of breaks, hence it seems likely that a large proportion of all breaks can serve as RNA chain initiation sites for core polymerase. Single-strand breaks in T7 DNA inhibit transcription by E. coli RNA polymerase holoenzyme. The inhibition is due to a decrease in the fraction of RNA polymerase holoenzyme molecules that can initiate a T7 RNA chain when DNA containing single-strand breaks serves as template. This decrease is probably accounted for by the finding that single-strand breaks serve as tight binding sites for RNA polymerase holoenzyme but that few such sites can serve as RNA chain initiation sites. It is concluded that the structural requirements for a “tight” binding site on DNA for RNA polymerase are less stringent than the requirements for an RNA chain initiation site. Thus, the fidelity of initiation of T7 RNA synthesis is governed not only through regulation of the sites on T7 DNA at which binding of RNA polymerase can occur, but also by what appear to be rigid structural requirements for RNA chain initiation by RNA polymerase once it has been bound.